229
Anal. Chem. 1985, 57. 229-234
two times) to be not useful for our particular purposes. The reaction of sugars with ABH resembles that reported by Lever (IO) for other hydrazides of aromatic and heterocyclic carboxylic acids. However, with p-hydroxybenzoic acid, which was found to give the best signal-to-blank characteristics (IO) of all reagents tested till now, we were able to obtain only a detection limit of about 0.5 pg for sucrose as compared to 15 ng for ABH. It could be speculated that a possible hydrogen bonding betweeen the amino group of the ABH reagent and hydroxyl groups of saccharides can positively affect the reaction or the spectral characteristics of the reaction products. The sensitivity of the recommended ABH system can be further improved. Among possible methods is the use of longer reaction times closer to the manually determined optimum, using segmented systems or an increased reaction temperature which would require the use of an adequate back pressure element after the photometric cell. Another alternative is the postcolumn supply of ABH to the detection system as a stable acidic or neutral solution and its mixing on the low-presure or high-pressure part of the reagent delivery system with a highly concentrated NaOH solution, whose concentration in the reaction mixture can then exceed the limits suggested in the present paper. The reagent discussed can be used in conjunction with a solid-phase catalytic reactor for the detection of both reducing saccharides and nonreducing saccharides, hydrolyzable to reducing subunits. The reagent has shown a good specificity for natural samples (plant extracts and wines), for which other detection principles (RI, direct UV photometry, UV photometry after derivatization) are not sufficiently specific to resolve sugars from accompanying interferences. ABH reagent was compared to other postcolumn derivatization reagents and was found to be for a given column packing/mobile phase combination the most specific for UV detection and sensitive with a detection limit of about 4 X lo+ mol of sucrose. It is reasonable to expect that ABH will be compatible with the borate anion-exchange separation method since a similar reagent (24) has been used under comparable reaction conditions in conjunction with the above mentioned separation method. One can also conclude, that the ABH reagent is
applicable to all the sugars investigated by Lever (IO) since a similar reaction mechanism can be expected.
ACKNOWLEDGMENT We are grateful to Kratos, Ltd., for the loan of a postcolumn reaction unit. M. W. F. Nielen is thanked for assistance in setting up the instruments. Registry No. BCA, 979-88-4; CAA, 107-91-5; TTB, 1871-22-3; HCF, 13746-66-2; AHB, 5351-17-7; raffinose, 512-69-6; sucrose, 57-50-1; glucose, 50-99-7; fructose, 57-48-7. LITERATURE CITED Green, J. 0. Natl. Cancer Inst. Monogr. 1988, 2 7 , 447. Kesler, P. B. Anal. Chem. 1987, 3 9 , 1416. Ohms, J. I.; Zeg, J.; Benson, J. V. Anal. Biochern. 1987, 2 0 , 51. Sinner, M.; Simatupang, M. H.; Dletrlchs, H. H. Wood Sci. Techno/. 1975, 9 , 307. (5) Frel, R. W. I n “Chemlcal Derlvatization in Analytical Chemlstry”; Frel, R. W., Lawrence, J. F., Eds.; Plenum: New York, 1981; Vol. I, pp
(1) (2) (3) (4)
2 11-340. (6) Lawrence, J. F.; Frel, R. W. “Chemical Derlvatizatlon In Llquld Chromatography”; Elsevier: Amsterdam, 1976. (7) Deekler, R. S.; Kroll, M. 0. F.; Beeren. A. J. B.; Van den Berg, J. H. M. J . Chromatogr. 1978, 749, 669. (6) Scholten, A. H. M. T. Thesis, Free University, Amsterdam, 1981. (9) Mopper, K.; Degens. E. T. Anal. Blochem. 1972, 4 5 , 147. (10) Lever, M. Anal. Biochern. 1982, 4 7 , 273. (11) Kldby, D. K.; Davldson, D. J. Anal. Blochem. 1973, 55, 321. (12) Sinner, M.; PUIS,J. J . Chromatogr. 1978, 756, 197. (13) Honda, S.; Matsuda, Y.; Takahashl, M.; Kakehi, K.; Ganno, S. Anal. Chem. 1980, 5 2 , 1079. (14) Honda, S.; Takahashl, M.; Kakehl, K.; Ganno, S. Anal. Biochem. 1981, 773, 130. (15) Honda, S.; Takahashi, M.; Nishimura. Y.; Kakehl, K.; Ganno, S. Anal. Blochem. 1981, 778, 162. (16) Kato, T.; Klnoshlta, T. Anal. Blochem. 1980, 706,238. (17) Grlmble, 0. K.; Barker, H. M.; Taylor, R. H. Anal. Biochem. 1983, 728, 422. (18) Nardln, P. Anal. Biochern. 1983, 737,492. P.; Ouhrabkovl, J.; Coplkovl, J. J. Chromatogr. 1980, 797 (19) Y!tng, 3 IO.
(20) Vrltnq, P.; Frei, R. W.; Brinkman, U. A. Th.; Nielen, M. W. F. J . Chro matogr. 1984, 295, 355. (21) Scobell, H. D.; Brobst, K. M.; Steele, E. M. Cereal Chem. 1977, 54 905.
(22) Kiatos HPLC Report, Carbohydrates, undated. (23) Davies, A. M. C.; Robinson, D. S.; Couchman, R. J . Chromatogr. 1974, 707,307.
RECEIVED for review March 26, 1984. Resubmitted and accepted September 4,1984.
7- [ (Chlorocarbonyl)methoxy1-4-methylcoumarin: A Novel Fluorescent Reagent for the Precolumn Derivatization of Hydroxy Compounds in Liquid Chromatography Karl-Erik Karlsson,l Donald Wiesler, Mark Alasandro, and Milos Novotny*
Department of Chemistry, Indiana University, Bloomington, Indiana 47405
A new reagent, 7-[(chlorocarbonyl)methoxy]-4-methyicoumarin, has been syntheslzed to form fluorescent derlvatives for the llquld chromatography of various hydroxy compounds. Reactlon condltlons were optlmlzed wlth model hydroxy sterolds and prostaglandins. Microcolumns of high chromatographlc efflclency were employed to resolve Isomeric compounds In both standard and blologlcal mixtures. On leave from the Department of Analytical Pharmaceutical Chemistry, Biomedical Center, University of Uppsala, Box 574 5-751 23 Uppsala, Sweden. 0003-2700/85/0357-0229$01.50/0
A sample derivatization step prior to chromatography has become a common part of numerous analytical determinations. In the area of gas chromatography (GC),such a step is primarily sought to improve volatility, thermal stability, and quantitation of polar compounds. In liquid chromatography (LC), the most frequent reason for sample derivatization is improving detection. As widely documented in the review literature (I-3), numerous precolumn reactions have now been successfully explored to enhance sensitivity and/or selectivity of chromatographic measurements. Formation of various fluorescent derivatives for LC de@ 1984 American Chemical Society
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ANALYTICAL CHEMISTRY, VOL. 57, NO. 1, JANUARY 1985
terminations has been popular in the cases where high-sensitivity measurements are essential. While a number of fluorescent "tagging" procedures are available (1-3) for acids, primary amines, or even carbonyl compounds, there has been a conspicuous lack of similar techniques for hydroxy compounds, a notable exception being the use of the l-ethoxy4-(dichloro-s-triazinyl)naphthalenereagent (4, Hydroxy compounds are ubiquitous in nature, with prostaglandins, steroids, and carbohydrates being representative examples of the important compound classes. In order to provide sensitive multicomponent measurements of such compounds in physiological fluids, GC has frequently been used (5) despite the frequent need for multiple derivatizations and potential problems of sample stability. Clearly, modern liquid chromatography offers certain advantages. In order to determine hydroxy compounds in various complex mixtures of biological importance at high sensitivity, a new fluorescence-yielding reagent has been developed in this work. 7-[(Chlorocarbonyl)methoxy]-4-methylcoumarin (CMMC) was prepared from an inexpensive commercially available precursor, 7-hydroxy-4-methylcoumarin, by treatment of its sodium salt with sodium bromoacetate followed by a reaction of the resulting aryloxyacetic acid with thionyl chloride. Optimum conditions for the reaction of CMMC with model steroids and prostaglandins were thoroughly investigated, while the reaction products were verified and characterized through their mass and fluorescence spectra. This new derivatization reaction was applied to complex synthetic and natural mixtures to assess its potential applications. Both the microcolumn and conventional LC were employed for the resolution of these derivatized sample components while measurement sensitivities were assessed.
EXPERIMENTAL SECTION Chemicals. 4-(Dimethylamino)pyridine,DMAP (Aldrich Chemical Co., Milwaukee, WI), employed as a catalyst, was purified by alumina column chromatography using diethyl ether as a starting material for the eluent. 7-Hydroxy-4-methylcoumarin, the synthesis of 74(chlorocarbonyl)methoxy]-4-methylcoumarin, CMMC, was received from the Eastman Kodak Co. Steroid standards were obtained from various commercial and private sources. The prostaglandin standards were obtained through the courtesy of John B. Landis, Upjohn Chemical Co., Kalamazoo, MI. Methylene chloride was dried by passing 20 mL through a 50 X 4 mm column filled with anhydrous sodium carbonate. A ll other chemicals were of analytical reagent grade. Synthesis. T o a solution made by neutralizing 13.09 g (94.2 mmol) of bromoacetic acid with 8.7 g (104 mmol) of sodium bicarbonate in 40 mL of water was added a solution made by dissolving 15.08 g (85.7 mmol) of 7-hydroxy-4-methylcoumarin in 85 mL of 1.1M aqueous sodium hydroxide. The mixture was heated under reflux for 1h; 12 M HCl was added dropwise until the intense yellow color vanished. Stirring was continued overnight. Acidification with HC1 yielded a pasty solid which was recrystallized from water, yielding 8.91 g (38.1 mmol, 44%) of white crystals, mp 208-211 "C. A solution of the acid in 57 g of freshly distilled thionyl chloride was heated under reflux for 1 h. Most of the excess thionyl chloride was removed by distillation and replaced with pentane. The resulting bluish green precipitate was recrystallized from ethyl acetate containing ca. 5% thionyl chloride and freed of solvent in vacuo, yielding 5.49 g of crystals (21.7 mmol, 57%) mp 129-130 "C. IR (KBr wafer): 1795, 1700, 1610, 1385, 1265, 1160, 1137, 915 cm-l. NMR (CDCI,): 6 7.69 (d, 1 H), 6.89 (dd, 2 H), 6.22 ( 8 , 1 H), 5.05 (s, 2 H), 2.41 (s, 3 H). Mass spectrum (70 eV), m / e 252 (M+, loo), 224 (39), 189 (53),159 (23), 147 (57). HRMS, m / e 252.0194 (calcd for Cl2H9C1O4,252.0189). Preparation of Standard Derivatives. Androsterone (0.09 g) was dissolved in 4 mL of dried methylene chloride. CMMC (0.081 g) and 1 mL of triethylamine were added. After 1h the organic phase was washed with 50 mL of 0.1 M phosphoric acid
and taken to dryness and the residue recrystallized twice from absolute ethanol. The pregnanediol derivative was prepared similarly by using a molar ratio of 1:2 (pregnanediol/CMMC). It was recrystallized twice by using absolute ethanol and once from methylene chloride/absolute ethanol (1:3). The purity of standard derivatives was checked by high-performance liquid chromatography (HPLC). ChromatographicEquipment. For the evaluation of reaction conditions, the following equipment was used. A Waters Assoc. Model 660 solvent programmer with Model 6000 pumps was used for evaluation of reaction conditions. Sample sizes of 10-20 pL were introduced via a Rheodyne Model 7125 injection valve. The detector was a Perkin-Elmer LC-55 spectrophotometer equipped with an 8-pL flow cell. The column used was a Microsorb C-18, 5 pm (Rainin Instrument Co., Inc., Emeryville, CA), 25 X 4.6 cm. The column effluent was monitored at 315 nm. The micro-HPLC system was comprised of a Varian 8500 syringe pump and a Valco Model C14W injection valve with an injection volume of 200 nL. The fluorescence detector was a Schoeffel FS 970 fluorometer equipped with a modified 160-nL flow cell. The excitation wavelength was 315 nm, while a 389-nm cutoff filter was used for emission measurements. Gradient elution was accomplished by using a homemade gradient mixing chamber (6)with a volume of 84 pL. The separation column used was made of a fused silica tube (1500 X 0.24 mm), slurry-packed with 3-pm Spherisorb ODS-2 as described elsewhere (7). Spectroscopic Measurements. A Model 5980A HewlettPackard dodecapole mass spectrometer was used for the identification of steroid derivatives, which were collected at the microcolumn's end and inserted by a direct-probe inlet into the mass spectrometer. Mass spectra from the separated biological compounds were compared with those obtained with derivatized standard steroids. Absorption spectra of the derivatives were recorded with a Perkin-Elmer Model 552 spectrophotometer, while fluorescence spectra were obtained by using an Aminco Bowman SPC. Evaluation of Reaction Conditions. Stock solutions of pregnanediol, androsterone, and a prostaglandin, PGF,,, were separately prepared by using methylene chloride as a solvent. CMMC and DMAP, in appropriate concentrations, were dissolved in dry methylene chloride. For the study of recoveries, a solution of the compound under study was transferred to a small glass vial and evaporated to dryness by using nitrogen gas. The residue was then dissolved in 100 pL of CMMC solution before adding DMAP. After thorough mixing, the reaction mixture was evaporated to dryness and the residue dissolved in the mobile-phase solution containing a known amount of anthracene or 1methylpyrene as an internal standard. The peak-height ratio of a derivative to the internal standard was determined and compared with the peak-height ratio given by a known amount of the derivatized steroid. Preparation of Plasma Sample. Fifty milliliters of pooled human plasma was analyzed using a modified version of the procedure described by Axelson and Sahlberg (8). Briefly, the procedure involved a fivefold dilution of plasma with water and heating it at 64 "C for 15 min, followed by passing the sample through a C-18 Sep-PAK (9) cartridge. The steroids were then eluted with methanol. Steroid sulfates were cleaved by solvolysis as previously described (IO). The sample was further purified using SP- and DEAE-Sephadex column chromatography to remove interfering acids and bases. The final eluant was evaporated to dryness with nitrogen. One hundred microliters of 0.023 M CMMC was added, followed by 15 pL of 0.1 M DMAP. Again, the reaction mixture was evaporated to dryness and the residue dissolved in 0.3 mL of acetonitrile. Afterward, the sample was transferred and mixed with 0.7 mL of water in a syringe connected to a C-18 Sep-PAK cartridge. Excess reagent and reaction byproducts were eluted with 20 mL of 30% acetonitrile/water. The derivatized steroids were recovered with 3 mL of acetonitrile, taken to dryness, and redissolved in 30 p L of 75% acetonitrile/water prior to LC.
RESULTS AND DISCUSSION Reaction Conditions. Acid chlorides are not widely employed in analytical derivatizations as reagents. Their 81.15ceptibility to hydrolysis to the parent acids makes them
ANALYTICAL CHEMISTRY, VOL. 57, NO. 1, JANUARY 1985 231
3 0
0
0.04
0. 02
concentration o f
0.06
0.08
0. 1
CMMC (M)
Flgure 1. Effect of CMMC on the reaction yield of the pregnanediol derivative: concentration of DMAP, 0.033 M; 7 pg of pregnanediol
used. Peak height ratio ( X ) 100
-
75
-
I
0
0.005
0.01
0.015
0.02
0.025
concentration o f catalyst (M)
Effect of the catalyst concentration on reaction yield: compounds derivatized, (0)pregnanediol and (0)PGF1,; concentration of CMMC, 0.02 M; samples, 7 pg of pregnanediol and 10 pg of PGF,,. Flgure 2.
difficult to maintain in a pure form. Moreover, such acids liberated by water present in the reaction mixture will decrease the concentration of the tertiary amine catalyst, usually required in acylation reactions, by ion-pair formation. Yet, if the water content in the reaction mixture can be kept low, offsetting advantages of the high reactivity of acid chlorides toward hydroxy functional groups are a short reaction time and a low reaction temperature. The latter is especially important when dealing with thermally labile compounds. In this study, methylene chloride, first passed through a short column containing anhydrous sodium carbonate (in order to decrease the water content), was chosen as a solvent for the reaction. In a first attempt to catalyze the reaction of CMMC with pregnanediol using pyridine, yields in the range of only 5-10% were observed. DMAP is a much more powerful catalyst, giving rate constants several orders of magnitude larger than pyridine (2). Indeed, when DMAP was used instead of pyridine, a rapid reaction took place to give a quantitative yield of a pregnanediol derivative. The yield data given in Figure 1 were obtained at ambient temperature; no time-dependent yield was ever observed. It can be seen from Figure 1 that a yield of 99% is reached when the CMMC concentration is approximately equal to that of DMAP. Consequently, it was of interest to find the optimum concentration of the catalyst with regard to the acid chloride concentration. Figure 2 shows that a yield of the pregnanediol derivative reaches a maximum value, which was found to be 102% ( n = 5). Therefore, by optimizing the concentration of DMAP relative to the concentration of CMMC, a quantitative yield can be obtained at lower reagent concentration (see Figure 1). When androsterone was used as a model
-
0
S
icc ~~
~
10
i s YIN
Flgure 3. Separation of derivatized prostaglandins: (1) PGA,; (2) PGE,; (3) PGF,,; mobile phase, acetonitrile/water/acetic acid (69:30:1); flow rate: 1.O mL/mln; spectrofluorometric detection.
compound at the optimum conditions found for pregnanediol, a yield of 100% (n = 3) was obtained. The reason for the decreased yield at higher DMAP concentrations is presently not known. The yield of the pregnanediol derivative was found to be quantitative in the range of 5-500 ng of underivatized steroid by using the optimized method. No attempt was made to further decrease the concentration of CMMC. To give additional illustration of the usefulness of CMMC as a derivatization agent, some prostaglandins were also tested. Just as was found for steroids, no time-dependent yield of the prostaglandin derivatives was noticed. As Figure 2 shows, the yield in this case is also decreased at higher DMAP concentrations, with the highest value obtained when the concentrations of CMMC and DMAP are equal. The absolute yield was not determined due to the limited amount of the prostaglandin available. However, when equimolar amounts of androsterone and PGAl (both react at only one position of their structures) were derivatized, they produced peak areas (UV detector) of comparable size, indicating a high yield even for the prostaglandin. Both derivatives have absorption maxima at 315 nm due to the presence of the coumarin moiety. Figure 3 shows a chromatogram of three derivatized prostaglandins. Comparison of the retention times of these derivatives in the reversed-phase system indicates that CMMC has reacted with all three hydroxy groups in PGF1, and with both grous in PGE1. Fluorescence Properties and Detection Limits. The
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ANALYTICAL CHEMISTRY, VOL. 57, NO. 1, JANUARY 1985
6
2
I , 0
i
1
HRS
Flgure 4. Separation of standard steroid derivatives: nonlinear gradient, 75-100% acetonltrile/water; initial flow rate, 1.O ML/min; (1) 1 lphydroxyandrosterone; (2)11/3+ydroxyetiocholanoIone; (3)tetrahydrocottisol; (4) &cottolone;(5) p-cortol; (6) a-cottolone, (7) a-cortol; (8) androsterone; (9) dehydroepiandrosterone; (10) pregnanetriol; (1 1) androstanediol; (12) pregnanediol.
Table I. Detection Characteristics of Steroid Derivatives compd derivatized
in mobile phase
detection limit, pg
androsterone
100
750
90
75
260 390
100
1100
90
616 1960
pregnanediol
% acetonitrile
75
fluorescence spectra of the pregnanediol derivative were recorded in acetonitrile/water mixtures ranging from 75 to 100% acetonitrile (Figure 4). The excitation maximum was at 320 nm and maximum emission at 380 nm (uncorrected values). As has been observed earlier, analytical reagents derived from the coumarin molecule give derivatives whose quantum yield is dependent on the composition of the solvent used ( I I , I 2 ) . This was also found to be the case with the CMMC esters of the steroids. There was no shift in excitation or emission spectra in the range of 75100% acetonitrile. However, a 75% decrease in the quantum yield was found with the nonaqueous solvent. The absorption spectra showed a maximum a t 315 nm which remained constant as did the molar extinction coefficients over the used solvent composition range. With microcolumn LC, the minimum detectable quantity (MDQ) for the androsterone derivative was 258 pg (90% acetonitrile, k’ = 2, n = 88 000). However, when the mobile phase was changed to 100% acetonitrile, the MDQ increased to 750 pg. The corresponding values for the pregnanediol derivative were 616 pg (k’ = 5.1, n = 72 700) and 1100 pg, respectively. To compare the detector response for different derivatives, the minimum detectable concentration (MDC) should be used. Table I shows that the MDQ will reach a minimum. This can be explained by changes in both detector response and changing peak widths at different capacity ratios. On the other hand, while comparing MDC values, it can be
no. of
MDC,
theoretical plates
mol/s x W2
87 500 88 000
2.9
125 000
0.5 0.012
67 600 72 700 120 500
0.46 0.012
3.0
seen that the detector response for the two derivatives is almost identical. However, the MDC seems to decrease by approximately 2 orders of magnitude when the mobile-phase composition is changed from 75% to 100% acetonitrile. This would be the case if the efficiency were constant irrespective of the capacity ratio. Going from 100% to 90% acetonitrile, the plate number for the androsterone derivative is virtually unchanged (87 500-88 000), but at 75%, it has increased to 125 100. This change in efficiency is probably due to some band broadening in the injector, the effect of which is more pronounced when a high acetonitrile content in the mobile phase is used. Applications. Ideally, an analytical reagent used for making fluorescent derivatives should be nonfluorescent or easily removable from the reaction mixture. The reagent used in this study is strongly fluorescent but can be hydrolyzed easily. If the reaction mixture is washed with a buffer solution, the corresponding fluorescent acid is formed. By selecting pH properly, the acid can be extracted into the aqueous phase. Distribution studies with this acid gave a value of 4.6for KD (the distribution constant) and pK6 equal to 2.8. This means that 99% of this acid can be removed by extracting the reaction mixture with an equal volume of a buffer with pH 5.5. When pH 6.0 was used, even the most polar compound in this study, the cortisol derivative, remained quantitatively in the organic phase. However, when this technique was used to
ANALYTICAL CHEMISTRY, VOL. 57, NO. 1, JANUARY 1985
233
it
I, b
1
2
3 HR8
Flgure 5. Separation of steroid sulfates isolated from human plasma. Experimental conditions are as in Figure 4: (1) 110-hydroxyetiocholanolone; (2) tetrahydrocortisol; (3) 0-cortolone; (4) androsterone; (5)dehydroepiandrosterone;(6) see text; (7) pregnanetriol; (8) pregnanediol; (9-1 1) see
text. clean up a derivatized plasma sample, it was found that even if the reagent itself was removed, many other disturbing peaks appeared in the chromatogram. Therefore, a modified solid-phase extraction procedure, as discussed previously, was adopted (13). There are numerous steroid metabolites commonly encountered in biological samples such as human blood or urine. To resolve such complex mixtures, highly efficient columns (more than 100 000 theoretical plates) will be required. An example of a high-resolution chromatogram is shown in Figure 5, featuring a metabolic profile of plasma steroids conjugated as sulfates prior to their solvolysis and derivatization. Tentative identification of steroids from this plasma sample was accomplished by comparing retention data with those of derivatized standards and, in some cases, through massspectral identification. While the early peaks in this chromatogram mostly represent the expected metabolites, the mass spectra of peaks 6 and 9-11 suggest some less usual androstane derivatives. It is of some interest that peaks 9-11 elute after the pregnanediol derivative of a greater molecular weight. Therefore, it appears possible that these compounds are actually polyhydroxylated androstanes with at least three or four derivatized hydroxy groups. A positive identification of all
metabolites may require availability of additional standards as well as the use of other structural techniques. It would not be surprising if new compounds are discovered, since the hitherto used gas-phase techniques may not be adequate for detection of more polar metabolites. Registry No. CMMC, 91454-65-8; PGA1, 14152-28-4;PGEI, 745-65-3; PGF1,, 745-62-0; lib-hydroxyandrostene, 57-61-4; lib-hydroxyetiocholanolone, 739-26-4;tetrahydrocortisol, 53-02-1; P-cortolone, 667-66-3; p-cortol, 667-65-2; a-cortolone, 516-42-7; a-cortol,516-38-1; androsterone, 53-41-8;dehydroepiandrosterone, 53-43-0; pregnanetriol, 27178-64-9; androstanediol, 571-20-0; 90-33-5; pregnanediol, 80-92-2; 7-hydroxy-4-methylcournarin, thionyl chloride, 7719-09-7.
LITERATURE CITED (1) Lawrence, J. F.; Frei, R. W. "Chemical Derivatization in Liquld Chromatography"; Elsevier: Amsterdam, 1976; Vol. 7. (2) Sternson, L. A. I n "Chemical Derivatization in Analytical Chemistry"; Frei, R. W., Lawrence, J. F., Eds.; Plenum Press: New York, 1981; Vol. 1, pp 127-203. (3) "Chemical Derivatization in Analytical Chemistry"; Frgi, R. W., Lawrence, J. F., Eds.; Plenum Pres$: New York, 1982; Vol. 2. (4) Chayen, R.; Gould, S.; Harell, A.; Stead, C. V. Anal. Biochem. 1971, 39,533-535. (5) Novotny, M.; Wiesler, D. I n "Separation Methods: New Comprehensive Biochemistry"; Deyl, Z.,Ed.; Elsevier: New York, 1984; Vol. 8, pp 41-147.
234
Anal. Chem. 1905, 57,234-237
(6) Karlsson, K.-E.; Novotny, M. HRC CC,J . High Resolut. Chromafogr. (7) (8) (9) (10) (11) (12)
Chromatogr. Commun. 1983, 7 , 411-413. Gluckman, J. C.; Hlrose, A.; McGuffin, V. L.; Novotny, M. Chromatooraahia 1983 f 7 ,. 303-309 - .- - - ' A x k o i , k-sahlberg, B. L. Anal. Len. 1981, 14, 771-782. Shackleton, C. H. L.; Whltney, J. 0. Clln. Chim. Acta 1980, 107, 231-243. Setchell. K. D. R.; Alme, B.; Axelson, M.; Sjovall, J. J. Steroid Biochem. 1978, 7 , 615-629. Lloyd, J. B. F. J . Chromatogr. 1979, 178, 249-258. Voeker, W.; Huber, R.; Zech, K. J. Chromatogr. 1981, 277, 491-507.
(13) Novotny, M.; Alasandro, M.; Konishi, M. Anal. Chem. 1983, 55, 2375-2377.
RECEIVED for review January 24, 1984. Resubmitted and accepted September 4, 1984. This work was sutmorted bv Grant PHS kOl GM 24349-05 from the Institute-& General Medical Scientes, US. Department of Health and Human Services.
Liquid Chromatographic Separation of Diastereomers and Structural Isomers on Cyclodextrin-Bonded Phases Daniel W. Armstrong,* Wade DeMond, and Ala Alak Department of Chemistry, Texas Tech University, Lubbock, Texas 79409
Willie L. Hinze and Terrence E. Riehl Department of Chemistry, Wake Forest University, Winston-Salem, North Carolina 27109
Khanh H. Bui Advanced Separation Technologies, Inc., 37 Leslie Court, P.O. Box 297, Whippany, New Jersey 07981
Elghty compounds are separated from thelr Isomers by LC uslng cyclodextrln-bonded columns. A varlety of structural Isomers (lncludlng polycycllc aromatlc hydrocarbons and prostaglandlns), geometrlc Isomers, and sterold epimers are examined. Cyclodextrln-bonded packlngs appear to be more widely applicable than elther normal or reversed-phase packlngs for these types of separatlons. Indeed, compounds that cannot be well resolved on more tradltlonal columns are oflen easily separated on thls statlonary phase. The separation mechanism ls based on lncluslon complex formatlon and Is responsible for the unusual but oflen predlctable selectlvltles observed.
The separation of isomeric compounds by liquid chromatography is an interesting but unevenly characterized field. The separation of enantiomers, for example, has received a considerable amount of attention from a number of researchers employing a variety of techniques (1-3). By comparison, the LC separation of diastereomers and structural isomers has been somewhat neglected. Although one can find isolated examples of specific separations of pairs of isomers, there are few broadly applicable approaches and, generally, little evaluation of the principles involved. One of the few techniques that has been reasonably well studied is that of argentation chromatography. Silica gel impregnated with silver ions has been shown to be selective for the separation of certain geometrical isomers such as cisand trans-retinols, pheromones, pesticides, and unsaturated fatty acids (4-7).Thallium ion has been used in place of silver for analogous separations (8). Geometrical isomers are, of course, only one subclass of diastereomeric compounds. Argentation chromatography is generally less successful in separating structural isomers, other types of diastereomers, and saturated compounds which are not able to form charge-transfer complexes. There is also the problem of a lack of reproducibility, high cost, and bleeding (due to the solubility of silver salts in many organic solvents).
An examination of the recent literature seems to indicate that the separation of structural and geometric isomers is more easily accomplished by normal-phase LC than by reversedphase LC. For example, silica gel is by far the preferred packing in the separation of isomers of retinal, retinol, and retinyl esters (9, 10). The separation of 0-,m-, and p-nitroaniline has been achieved with near-base-line resolution on an alumina column but not by reversed-phase LC (11). One class of isomeric compounds (i.e., diastereomers) where reversed-phase LC seems to have had as much success as normal-phase LC is in the separation of steroid epimers (12,13). It has been noted that some steroid epimers are best separated by normal-phase LC and others by reversed-phase LC, and some are not well separated by either technique (12-15). Often recycling is needed to achieve these separations, particularly when the epimeric center is ?Czo of the steroid structure (12). It is apparent that in some cases the separation of a pair of isomers is facile (syn- and anti-azobenzene, for example) while in other cases very specific conditions are required. There are also several examples of intractable isomers for which no really effective LC technique currently exists. For example, the separation of all four epimers of estriol (i.e., estriol, 16-epiestriol, 17-epiestriol, and 16J7-epiestriol) has not been reported to our knowledge (13). Benzo[a]pyrene and benzo[e]pyrene (as well as some other polycyclic aromatic hydrocarbons) are known to be difficult to separate, particularly in complex mixtures (16-18). It is also apparent that there is no single packing or procedure that can be considered widely applicable for the LC separation of diastereomers and structural isomers. Certainly the development of an effective packing that can discriminate between a variety of similar compounds on the basis of their geometry or orientation and that would allow one to avoid using specially prepared packings, multiple columns, and recycling, would be useful in LC. It will be demonstrated in this report that the cyclodextrin column is particularly adept at separating both diastereomers and structural isomers. This is because the separation is largely due to inclusion complex formation which provides
0003-2700/85/0357-0234$01.50/00 1984 American Chemical Society
