60

STRUCTURE AND ANALYSIS

[5]

[5] H i g h - P e r f o r m a n c e L i q u i d C h r o m a t o g r a p h y of Retinoid Isomers: An Overview

By C. D. B. BRIDGES Introduction

The separation and quantitation of different retinoid isomers is essential in studies of retinoids in the visual systems of vertebrates and invertebrates, in investigations of retinoid isomer interconversions in other tissues (e.g., retinoic acid in rat liver), and in determining the biopotency of vitamin A in foodstuffs and food supplements such as fish liver oils. In nature, vitamin A compounds can exist as the aldehyde, ester, or acid forms of retinol, 3-hydroxyretinol, or 3,4-didehydroretinol. Additionally, the aldehydes are often derivatized with hydroxylamine in the tissue and extracted in the form of the corresponding oximes. In practice, the separation of the various mono-cis isomers is comparatively easy provided only a single class of retinoid derivatives is under study. More usually, however, several retinoid classes of widely differing polarities are present in the mixture being investigated. If these classes include retinyl esters and retinols, the researcher may attempt the separation in a single injection, usually employing a step gradient. The disadvantage of this approach is the long time it takes the column to reequilibrate to low-polarity eluting conditions after the final separating step that utilizes a high-polarity eluent mixture. An alternative approach is to carry out all the retinyl ester separations in a series of injections under low-polarity, isocratic conditions, then change the mobile phase to an appropriately more polar mixture of solvents and separate the retinol isomers in a second series of injections. Extraction Methods

Methods of retinoid extraction are fairly straightforward in most tissues, and have been described previously. 1,2 However, special procedures must be used to extract retinoids in their native isomeric configuration from visual pigments. This is because the simple use of a denaturing G. W. T. Groenendijk, P. A. A., Jansen, S. L. Bonting, and F. J. M. Daemen, this series, Vol. 67, p. 203. 2 C. D. B. Bridges and R. A. Alvarez, this series, Vol. 81, p. 463.

METHODS IN ENZYMOLOGY, VOL. 189

Copyright © 1990 by Academic Press, Inc. All rights of reproduction in any form reserved.

[5]

OVERVIEW OF H P L C OF RETINOID ISOMERS

61

solvent such as methanol (even under red or gold light) causes the 11-cisretinal prosthetic group to isomerize to other mono-cis (notably the 13cis) and all-trans isomers. The method devised by Groenendijk et al. 3 for photoreceptor membranes or detergent-solubilized material overcame this problem by carrying out the methanol denaturation in the presence of a 1000-fold molar excess of hydroxylamine. At these concentrations (10200 mM), oxime formation was so fast that it prevented isomerization of the liberated retinal. This method, or variations of it, has been applied to visual pigment systems based on retinal, 3,4-didehydroretinal, and 3hydroxyretinal.4-7 The procedure 3 is to add a 1000-fold molar excess of 1 M hydroxylamine (pH 6.5) to 10-50/zM of visual pigment in a membrane suspension. Methanol is added to a concentration of 70% (v/v), then water and dichloromethane are added until the three constituents are present in equal volumes. After vortexing, the mixture is centrifuged at 10,000 g for 1 min and the lower layer collected with a syringe. The procedure is repeated 3 times, giving a recovery of 93 - 7%. Suzuki et al. 8 used a conceptually similar method that depended on using formaldehyde as a protective agent, perhaps by blocking amino groups on protein and lipid. Pepe and Schwemer 9 described a method based on that of Pilkiewicz et al.l° Retinals are extracted from rod outer segment preparations in digitonin by adding sodium dodecyl sulfate to 1% final concentration, incubating for l0 min at 22°, then stirring with dichloromethane-methanol (1 : l, v/v) at 4 °. The resulting emulsion is centrifuged, and the lower dichloromethane layer containing the retinal isomers is withdrawn. The retinals are transferred to hexane and analyzed on a Spherisorb ODS 5/xm column used in normal phase with 10% ether in hexane. This column has the advantage that contamination of the sample with detergent and water does not influence resolution. The authors note that there is variability in performance between columns, probably owing to incomplete coverage of the packing material by chloroalkylsilanes. 3 G. W. T. Groenendijk, W. J. de Grip, and F. J. M. Daemen, Biochem. Biophys. Acta 617, 430 (1980). 4 T. H. Goldsmith, B. C. Marks, and G. D. Bernard, Vision Res. 26, 1763 (1986). 5 T. Suzuki and M. Makino-Tasaka, Anal. Biochem. 129, I II (1983). 6 T. Seki, S. Fujishita, M. Ito, N. Matsuoka, C. Kobayashi, and K. Tsukida, Vision Res. 26, 255 (1986). 7 T. Seki, S. Fujishita, M. Ito, N. Matsuoka, and K. Tsukida, Exp. Biol. 47, 95 (1987). 8 T. Suzuki, Y. Fujita, Y. Noda, and S. Miyata, Vision Res. 26, 425 (1986). 9 I. M. Pepe and J. Schwemer, Photochem. Photobiol. 45, 679 (1987). i0 F. G. Pilkiewicz, M. J. Pettei, A. P. Yudd, and K. Nakanishi, Exp. Eye Res. 24, 421 0977).

62

STRUCTUREAND ANALYSIS

[5] 4

A

00 32S

0"°11

0

~0 m|n.

J

FIG. 1. Separation of isomers of retinyl stearate. Peak 1, 13-cis-; Peak 2, 1l-cis-; Peak 3, 9-cis-; Peak 4, all-trans-retinyl stearate. Column, Ultrasphere 5 tzm (250 x 4.6 mm, 15,000 minimum plate count); mobile phase, diethyl ether (0.4%)/n-hexane at 0.7 ml/min; detection at 325 nm. [From R. A. Alvarez, S. L. Fong, and C. D. B. Bridges, Invest. Ophthalmol. Visual Sci. 20, 304 (1981).]

High-Performance Liquid Chromatographic Techniques It is usually easy to develop a highly efficient method of separating the isomers of a particular class of retinoids. F o r most retinoid classes, with the exception o f retinoic a c i d , normal-phase systems have been used almost exclusively. Figure 1 illustrates the separation o f 13-cis-, l l-cis-, 9-cis-, and all-trans-retinyl stearates. H Figure 2 shows the separation o f a mixture o f retinal isomers obtained by irradiation of all-trans-retinal in ethanol: the mixture contains about 22% 13-cis-, 29% 11-cis-, 11% 9-cis-, ]l R. A. Alvarez, S.-L. Fong, and C. D. B. Bridges, Invest. Ophthalmol. Visual Sci. 20, 304 (1981).

[5]

OVERVIEW OF H P L C OF RETINOID ISOMERS

I

0

I

I

10

I 20

63

I

time (min) FIG. 2. Separation of isomers of retinal in a mixture generated by irradiation of a solution of aU-trans-retinal in ethanol. Peak 13, 13-cis-; Peak ll, l l-cis-; Peak 9, 9-cis-; Peak 7, 7-cis-; Peak AT, all-trans-retinal. Column, /zPorasil l0 wm (3026 minimum plate count); mobile phase, diethyl ether (12.5%)/n-hexane at 0.6 ml/min; detection at 310 nm. [From C. D. B. Bridges, S.-L. Fong, G. I. Liou, R. A. Alvarez, and R. A. Landers, in "Progress in Retinal Research" (N. N. Osborne and G. J. Chader, eds.), p. 137. Pergamon, Oxford, 1983.] 1% 7 - c i s - , a n d 37% a l l - t r a n s - r e t i n a l ( m i n o r a m o u n t s o f d i - c i s i s o m e r s m a y b e p r e s e n t , b u t t h e y a r e n o t r e s o l v e d ) . ~z N o r m a l - p h a s e s y s t e m s h a v e a l s o b e e n m o s t u s e f u l in s e p a r a t i n g t h e i s o m e r s p r e s e n t in a m i x t u r e o f d i f f e r e n t c l a s s e s o f r e t i n o i d s . F i g u r e 3 shows an example of the separation of a variety of retinoid isomers ranging in p o l a r i t y f r o m 1 1 - c i s - r e t i n y l p a l m i t a t e to a l l - t r a n s - 3 - d e h y d r o r e t i n o l . I n this i n s t a n c e , a s t e p - g r a d i e n t e l u t i o n m o d e w a s u s e d , s t a r t i n g w i t h 0 . 2 % d i o x a n e in n - h e x a n e a n d finishing w i t h 12% d i o x a n e in n - h e x a n e . 13 I n F i g . 4, a q u a t e r n a r y m i x t u r e o f s o l v e n t s w a s u s e d in t h e i s o c r a t i c m o d e to 12C. D. B. Bridges, S.-L. Fong, G. I. Liou, R. A. Alvarez, and R. A. Landers, in "Progress in Retinal Research" (N. N. Osborne and G. J. Chader, eds.), p. 137. Pergamon, Oxford, 1983. 13C. D. B. Bridges, S.-L. Fong, and R. A. Alvarez, Vision Res. 20, 355 (1980).

64

STRUCTURE AND ANALYSIS

[5] Io

5 13o~4

2,3

I

9 6

O0 340 OO'31

7' I

-~ O

15

! o

~n

.I 2o

FIG. 3. Step-gradient separation of retinyl esters, retinals, retinal o×ime, retinols, and 3dehydroretinol. Peak 1, 1l-cis-retinyl palmitate; Peak 2, aU-trans-retinyl stearate; Peak 3, all-trans-retinyl palmitate; Peak 4, all-trans-retinyl oleate; Peak 5, all-trans-retinyl acetate; Peak 6, 13-cis-retinal; Peak 7, 11-cis-retinal; Peak 8, 9-cis-retinal; Peak 9, all-trans-retinal; Peak 10, all-tram-retinal oxime (syn conformer); Peak 11, 13-cis-retinol; Peak 12, 11-cisretinol; Peak 13, 9-cis-retinol; Peak 14, all-trans-retinol; Peak 15, all-trans-3-dehydroretinol. Column, Lichrosorb 10 ~m; mobile phase, dioxane/n-hexane run as follows: 0.2% dioxane for 21 min, 2% for 27 min, 12% for 25 min; flow rate, 1 ml/min; detection at 340 nm. [From C. 13. B. Bridges, S. L. Fong, and R. A. Alvarez, Vision Res. 20, 355 (1980).1

obtain excellent separation of retinal, retinal oxime (syn and anti conformers), and retinol isomers.~4 A summary of normal-phase systems for separating retinoid isomers is given in Table I. The major component of the mobile phase is nearly always n-hexane, although other solvents have been sporadically used (e.g., benzene, pentane, heptane, and octane have been used occasionally by the authors). When retinyl ester isomers are being separated, a small t4 G. M. Landers and J. A. Olson, J. Chromatogr. 438, 383 (1988).

[5]

OVERVIEW OF H P L C OF RETINOID ISOMERS

65

~'N RETtO& OXlME

0.1~

~ 1 1 nE'rlH~ oxit~ r~MEnS O

DENSITY

I

A i'-

O

BC

II

IEF

I

G

J'

o ~'I1ME,

IN ~

FIG. 4. Separation of retinol, retinal, and retinal oxime isomers using a quaternary mixture as the mobile phase. Peak 1, 13-cis-retinal; Peak 2, ll-cis-retinal; Peak 3, 9-cisretinal; Peak 4, 7-cis*retinal; Peak 5, all-trans-retinal; Peak 6, syn-1 l-cis-retinal oxime; Peak 7, syn-ail-trans-retinal oxime; Peak 8, syn-9-cis-retinal oxime + syn-13-cis-retinal oxime; Peak 9, anti- 13-cis-retinal oxime; Peak 10, anti- 1l-cis-retinal oxime; Peak 11, 11-cis-retinol; Peak 12, 13-cis-retinol; Peak 13, anti-9-cis-retinal oxime; Peak 14, 9,1 l-cis-retinoi; Peak 15, anti-all-trans-retinal oxime; Peak 16, 9-cis-retinol; Peak 17, ail-trans-retinol. The letters refer to the retention times for the following compounds: A, syn-9,1 l-cis-retinal oxime; B, syn-7-cis-retinal oxime; C, syn-9,13-di-cis-retinal oxime; D, anti-9,13-cis-retinai oxime; E, anti-9,1 l-cis-retinal oxime; F, anti-7-cis-retinal oxime; G, 7-cis-retinol. Column, two Lichrosorb Si 60 5/~m (250 z 4 mm) in series; mobile phase, ethyl acetate (11.2%)/dioxane (2%)/ l-octanol (1.4%)/n-hexane; flow rate, 1 ml/min; detection at 325 nm. [From G. M. Landers and J. A. Olson, J. Chromatogr. 438, 383 (1988).]

proportion of diethyl ether or dioxane is added as a polar modifier to the mobile phase, whereas for the more polar retinoids such as the retinals and retinols larger proportions are employed. The use of ternary or even quaternary mixtures has become more common in recent work, particularly where the isomers of different retinoid classes are being separated from the same mixture. In general, reversed-phase columns have not been used to separate the isomers of any retinoid class except retinoic acid (Table II). A notable recent exception has been the successful separation of mono- and di-cis-

66

STRUCTURE AND ANALYSIS

[5]

TABLE I NORMAL-PHASE SYSTEMS USED FOR SEPARATING RETINOID ISOMERS Mobile p h a s e (%) Retinoid °

(1) b

(2)

(3)

(4)

B, D, F

h

--

--

Lithosorb 5 / ~ m

c

B, G

h

--

Si-60 (Merck) 5 / ~ m

d

B, C, G

h

--

Si-60 (Merck) 5 / x m

e

B-E

h h

L i c h r o s o r b Si-60 (two in series) Zorbax SIL 7 / x m

f

A-C, G C

h

--

Spherisorb 5 / x m ODS

h

B, D, F

b

--

YMC-PACK 3/~m

i

B, D, F

h

--

YMC-PACK 3/~m

i

B, D, G

h

--

Sorbax SIL 7 a m

j

C, D

h

--

Lichrosorb 5 / z m

k

B

h

--

--

h

--

--

C, F

h

--

m

C, D, G

h

--

Z o r b a x S1L

n

A-D, G

h

Ethanol (0.15-0.05) Ethanol (0.05-0.075) --

Partisil 10-ODS and Zorbax C N 5 / ~ m Partisil 10-ODS and Zorbax C N 5 / z m Lichrosorb Si-60 5 / x m

1

B

--

h

--

--

D

h

--

--

A

h

--

--

L i c h r o s o r b 10/zm or /tPorasil L i c h r o s o r b 10/~m or /xPorasil MicroPak Si-5 or L i c h r o s o r b Si-60-5 Supelcosil LC-Si 3 / ~ m or Ultrasphere Si-60 5 / x m

o

A-D, G

B

h

--

--

A

h

Dioxane (14-18) l-Octanol (3.8) Dioxane (l.0) Ethyl acetate (I 1.2) Diethyl e t h e r (1-7) Diethyl e t h e r (10) tert-Butyl methyl ether (1-6) Diethyl e t h e r (8) Diethyl e t h e r (7) Diethyl e t h e r (5) 2-Octanol (1) Dioxane (5) Diethyl e t h e r (15-50) Diethyl e t h e r (6-7) Diethyl e t h e r (0.4-20) Dioxane (0.2-12) Dioxane (6) tert-Butyl methyl e t h e r (0.2-0.5) Dioxane (4-5) Diethyl ether (0.4)

--

--

2-Propanol (0.2) 2-Propanol (0.1) Dioxane (2.0) --Ethanol (0.12-1. l) Ethanol (0.08) Ethanol (0.075) --

l-Octanol (1.4) --

Column

Ultrasphere Si-60 5 / z m and Spherisorb C N 5 # m

Ref.

g

!

o p q, r

s

[5]

OVERVIEW OF H P L C OF RETINOID ISOMERS

67

TABLE I (continued) Mobile phase (%) Retinoid a

(1) b

B, C

h

C, G

h

(2) Dioxane (9) Diethyl ether (6-12)

(3) Diethyl ether (0.4) --

(4)

Column

p~Porasil

Ref.

t

a A, Retinyl esters; B, retinol; C, retinal; D, retinal oxime; E, retinoic acid; F, hydroxyretinol and derivatives; G, 3-dehydroretinol and derivatives. b The major component (1) of the eluent is nearly always n-hexane (h); benzene (b) was used in one instance. One of the authors has also used n-pentane, n-heptane, n-octane, and isooctane. c T. H. Goldsmith, B. C. Marks, and G. D. Bernard, Vision Res. 26, 1763 (1986). d B. Stancher and F. Zonta, J. Chromatogr. 312, 423 (1984). e F. Zonta and B. Stancher, J. Chromatogr. 3t11, 65 (1984). G. M. Landers and J. A. Olson, J. Chromatogr. 438, 383 (1988). g T. Suzuki, Y. Maeda, Y. Toh, and E. Eguchi, Vision Res. 28, 1061 (1988). h I. M. Pepe and J. Schwemer, Photochem. Photobiol. 45, 679 (1987). i T. Seki, S. Fujishita, M. Ito, N. Matsuoka, and K. Tsukida, Exp. Biol. 47 (1987). J T. Suzuki and M. Makino-Tasaka, Anal. Biochem. 129, 11 ! (1983). k y. Shichida, K. Nakamura, T. Yoshizawa, A. Trehan, M. Denny, and R. S. H. Liu, Biochemistry 27, 6495 (1988). t p. V. Bhat and A. Lacroix, this series, Vol. 123, p. 75. m T. Seki, S. Fujishita, M. Ito, N. Matsuoka, C. Kobayashi, ad K. Tsukida, Vision Res. 26, 255 (1986). n T. Suzuki, Y. Fujita, Y. Noda, and S. Miyata, Vision Res. 26, 425 (1986). o C. D. B. Bridges, S.-L. Fong, and R. A. Alvarez, Vision Res. 20, 355 (1980). P G. W. T. Groenendijk, W. J. de Grip, and F. J. M. Daemen, Biochim. Biophys. Acta 617 (1980). q C. D. Bridges and R. A. Alvarez, Science 236, 1678 (1987). ' C. D. B. Bridges, R. A. Aivarez, S.-L. Fong, G. I. Liou, and R. J. Ulshafer, Invest. Ophthalmol. Visual Sci. 28, 613 (1987). C. D. B. Bridges, R. A. Alvarez, S.-L. Fong, F. Gonzalez-Fernandez, D. M. K. Lam, and G. 1. Liou, Vision Res. 24, 1581 (1984). ' K. Tsukida, R. Masahara, and M. Ito, J. Chromatogr. 192, 395 (1980). r e t i n o l s w i t h a c o l u m n p a c k e d w i t h w i d e - p o r e (300 ,~) s i l i c a b o n d e d t o a p o l y m e r i c Cl8 s t a t i o n a r y p h a s e ( F i g . 5). 15

High-Performance Liquid Chromatograms Retinoid Isomers

of Radiolabeled

I n c e r t a i n m e t a b o l i c s t u d i e s it is o f t e n n e c e s s a r y t o o b t a i n a n a c c u r a t e r a d i o a c t i v e e l u t i o n p r o f i l e . T h i s c a n b e a p r o b l e m if t h e i s o m e r o f i n t e r e s t t5 W. A, MacCrehan and E. Schonberger, J. Chromatogr. 417, 65 (1987).

68

STRUCTURE AND ANALYSIS

[5]

TABLE II REVERSED-PHASE SYSTEMS FOR SEPARATING RETINOID ISOMERS

Retinoid Retinol

Retinoic acid Retinoic acid

Mobile phase Methanol-n-butanol-water (65 : l0 : 25, v/v/v) 10 mM ammonium acetate (pH 3.2) Methanol-water (75 : 25, v/v), l0 mM ammonium acetate Acetonitrile-dichloromethane (90 : 10) 10 mM acetic acid

Column

Ref.

Vydac 201 TP, C~8, 5/zm

a

Ultrasphere XL ODS, 3 p.m or Zorbax ODS, 5/zm Zorbax NH2, 5/.~m

b c

a W. A. MacCrehan and E. Schonberger, J. Chromatogr. 417, 65 (1987). b R. W. Curley, Jr., and J. W. Fowble, Photochem. Photobiol. 47 (1988). P. V. Bhat and A. Lacroix, this series, Vol. 123, p. 75.

2

A 20

30 rain FIG. 5. Separation of retinol isomers by reversed-phase high-performance liquid chromatography. Peak 1, di-cis-; Peak 2, ll-cis-; Peak 3, 9-cis-; Peak 4, 13-cis-; Peak 5, all-transretinol. Column, Vydac 201 TP 5/.~mC~8(250 x 4.6 mm); mobile phase, methanol~n-butanol~ water (65 : 10 : 25) containing 10 mM ammonium acetate (pH 3.2) at l ml/min; detection at 325 nm. [From W. A. MacCrehan and E. Schonberger, J. Chromatogr. 417, 65 (1987).]

e l u t e s w i t h i n a short t i m e o f a n o t h e r . A n e x a m p l e is 11-cis-retinol, w h i c h is g e n e r a t e d e n z y m a t i c a l l y w h e n all-trans-retinol is i n c u b a t e d with hom o g e n a t e s o f o c u l a r p i g m e n t e p i t h e l i u m . 16,17 H o w e v e r , its p e a k is c l o s e l y f o l l o w e d b y that o f 13-cis-retinol, a n i s o m e r that is f o r m e d n o n s p e c i f i cally, e v e n in h o m o g e n a t e s f r o m t i s s u e s l a c k i n g the i s o m e r a s e (e.g., b r a i n , liver) o r w h e n the e n z y m e itself has b e e n i n a c t i v a t e d b y heat,

16C. D. Bridges and R. A. Alvarez, Science 236, 1678 (1987). t7 p. S. Bernstein, W. C. Law, and R. R. Rando, Proc. Natl. Acad. Sci. U.S.A. 84, 1849 (1987).

[5]

OVERVIEW OF H P L C OF RETINOID ISOMERS

69

15

co I

oT - "

11

x

AT I

E "o

i

20

i

rain

i

40

FIG. 6. Separation of radiolabeled retinol isomers extracted from a homogenate of pigment epithelium-choroid incubated with all-trans-[l 1,12JH]retinol [conditions as described by C. D. B. Bridges and R. A. Alvarez, Science 236, 1678 (1987)]. Peak 11 (shaded), ll-cisretinol; Peak 13, 13-cis-retinol; Peak AT, aU-trans-retinol; the small peak on the rising phase of the AT peak is probably 9-cis-retinol, but the other peaks have not been identified. Column, Supelcosil 3/~m (150 x 4.6 mm); mobile phase, dioxane (4%)/n-hexane at 0.6 ml/ min; fractions were manually collected and counted on a Packard Tricarb liquid scintillation counter.

trypsin, or phenymethylsulfonyl fluoride (PMSF). 16 In order to resolve unequivocally and clearly the radioactivities associated with these two isomers when they are generated from tritiated all-trans-retinol, it has been found necessary to count fractions that have been collected at 0. lmin intervals. This procedure is most reliably carried out manually. 16 A typical radioactive profile derived by this approach is illustrated in Fig. 6. More recently, comparatively acceptable results have been obtained in the authors' laboratory with a carefully adjusted " F o x y " fraction collector (Isco, Lincoln, NE).