958
Anal. Chem. 1984, 56,958-962
Synthesis and Liquid Chromatographic Evaluation of Some Chiral Derivatizing Agents for Resolution of Amine Enantiomers C. Randall Clark* and Jeffrey M. Barksdale' Division of Medicinal Chemistry, Department of Pharmacal Sciences, School of Pharmacy, Auburn University, Auburn, Alabama 36849
A serles of 1-[(4-substituted-phenyi)sulfonyl]prolylchlorides were syntheslzed and evaluated as chiral derlvatlzing agents for the ilquld chromatographic analysis of enantiomeric amines. The dlastereomerlc l{( 4-nitrophenyl)suifonyi]proiinamides showed strong UV absorbance propertles and were separable by both normal-phase and reversed-phase liquid chromatographic technlques.
The reported methods for the chromatographic resolution of enantiomers fall into three categories. The first makes use of the differences in rates of interaction of enantiomers with chiral stationary materials. Kotake et al. (I) reported the chromatographic resolution of amino acid enantiomers utilizing a stationary phase of cellulose. Since this initial report much effort has been put forth in studying the GC resolution of enantiomers on chiral stationary phases and numerous such stationary phases have been described (2,3). More recently, the liquid chromatographic separation of enantiomers on chiral stationary phases has enjoyed a great deal of attention. These stationary phases include: chiral ligands bonded to silica (4) or Sephadex (5),chiral crown ethers (6),and microcrystalline cellulose triacetate (7). The second chromatographic resolution method involves the conversion of the enantiomers to diastereomers by reaction with a chiral derivatizing agent. The resulting diastereomers may be separated on a chiral or an achiral stationary phase. Two different mechanisms have been postulated for this separability. One involves differences in molecular structure and polarity of the diastereomers (8). The other involves differences in energies of adsorption of the diastereomers (9). This technique was reported by Stoll and Hofmann (10) in 1938 and both GC (2,3)and LC ( 3 , I I )have been utilized in this capacity. A third, more recent, method for liquid chromatographic resolution utilizes an achiral stationary phase and a mobile phase which contains a chiral eluent. The recently described (12) separation of enantiomeric and diastereoisomeric mixtures of ephedrine and pseudoephedrine using nickel dithiocarbamate complexes is an example of this type of procedure. Drug enantiomers often have different biological activities; thus their differentiation is an issue of medical and regulatory importance (13). This importance is illustrated in cases where one enantiomer is under regulatory control while the other is not. In addition, the enantiomeric makeup of a sample may give an indication as to its origin (14). Hufsey and Cooper (15) report that certain drugs derived from a natural source usually exist in one enantiomeric form while the synthesized counterpart is usually a mixture of enantiomers. The procedure reported herein utilizes the high separation efficiency of HPLC to resolve diastereomeric amides. These amides were formed by the reaction of the enantiomeric amines with a chiral derivatizing agent. The chiral derivatizing Present address: Medical Technology Program, Auburn University at Montgomery, Montgomery, AL 36193. 0003-2700/84/0356-0958$0 ISO10
agent had a 2-fold purpose. Its primary use was to add a second chiral center, thus forming the diastereomeric products. Futhermore, a chromophoric group was introduced into the chiral derivatizing agent in order to enhance the ultraviolet absorption characteristics of the amines.
EXPERIMENTAL SECTION General Procedure. Melting points were determined on a Thomas-Hoover melting point apparatus in open capillary tubes and are uncorrected. NMR spectra were determined in deuteriochloroform with tetramethylsilane as the internal standard on a Varian T-6OA spectrometer. Ultraviolet absorption spectra were determined in absolute ethanol with a Perkin-Elmer 200 UV spectrophotometer. All reagents and chemicals were of reagent grade quality of better and were used as purchased without further purification. The phenylsulfonyl chlorides, L-(-)-proline, and dand l-a-methylbenzylaminewere obtained from Aldrich Chemical Co., Milwaukee, Wi. Samples of d- and &pseudoephedrine, dand l-ephedrine,and d- and 1-amphetaminesulfate were obtained from Sigma Chemical Co., St. Louis, MO. HPLC grade n-heptane, acetonitrile, methanol, and tetrahydrofuran were obtained from Fisher Scientific Co., Fair Lawn, NJ. The water used in the chromatographic mobile phase was double distilled and pumped through a chromatographic guard column (7 cm X 2.1 cm i.d.) dry packed with Whatman C0:PELL ODS (30-38 wm) and equipped with 2-pm frits. Synthesis of (Phenylsulfony1)prolines. A solution of L(-)-proline (0.040-0.045 mol) in 40 mL of tetrahydrofuran and 200 mL of 10% (w/v) potassium carbonate was placed in a three-necked flask equipped with a magnetic stirrer, addition funnel, reflux condenser, and heating mantle. A solution of the appropriate acid chloride (0.037-0.043 mol) in 40 mL of tetrahydrofuran was added in a dropwise fashion. The resulting mixture was heated at approximately 50 O C for 3 h and maintained at pH 8 or above. The mixture was cooled, acidified to pH 2, and extracted with chloroform. The product was further purified by extraction into aqueous potassium carbonate then back into chloroform. The chloroform extracts were dried and evaporated and the resulting residue was recrystallized from petroleum ether and benzene. Synthesis of (Phenylsulfony1)prolylChlorides. A sample of the appropriate (phenylsulfony1)proline(approximately 0.015 mol) in 100 mL of benzene was placed in a three-necked flask equipped with a magnetic stirrer, addition funnel, reflux condenser, drying tube containing anhydrous calcium sulfate, and a heating mantle. A solution of thionyl chloride (&fold molar excess) in 50 mL of benzene was added to the mixture in a dropwise manner. The reaction mixture was heated at 35 to 40 "C until the formation of the acid chloride was complete (usually 48 h). The progress of the reaction was monitored by infrared spectrophotometry. Upon completion, the benzene and excess thionyl chloride were evaporated, and the resulting residue was placed under nitrogen. The residues consisted of viscous oils in all cases except 1-[(4-nitrophenyl)sulfonyl]prolylchloride, a crystalline solid which was purified by recrystallization using HPLC grade n-heptane. Anal. Calcd for 1-[(4-nitropheny1)sulfonyl]prolylchloride (mp 110-110.5 'C): C, 41.44; H, 3.45; N, 8.79. Found: C, 41.50; H, 3.48, N, 8.76. Synthesis of Amide Diastereomers. A solution of the enantiomeric amine (0.013-0.015 mol) in 40 mL of tetrahydrofuran and 150 mL of 10% (w/v) potassium carbonate was placed in a three-necked flask equipped with a magnetic stirrer, addition funnel, reflux condenser, and heating mantle. A solution of the 0 1984 American Chemical Society
ANALYTICAL CHEMISTRY, VOL. 56, NO. 6, MAY 1984
959
Table I. physical Properties of Model Amide Diastereomers X
O
I
-0 N
g C II - NI O
X
diastereomeric form
H
1; ‘0 UV properties
IR absorbances: cm-’
Q,
mp, “C
N-H
C=O
so,
136-138 106-108 153-155 127-129 116.5-1 18.5 105-109 189-191 163-165
3400 3400 3402 3405 3400 3400 3405 3405
1662 1662 1665 1665 1664 1665 1670 1670
1350, 1157 1350,1155 1355,1160 1355,1158 1348,1152 1350,1152 1350,1160 1350,1158
nm
E maxC
13 400 f 1 2 500 18 100 i 17 500 i 8 950 f 9 250 i
236 2 36 242.5 242.5 262 262
+_
3210i 1520 t 4120 t 3410 f 8140 i 7560 t 8750 i 9640 t
331 574 1880 2120 215 490
UV spectra obtained in absolute ethanol. a Solution data obtained in spectrophotometric grade CHCl,. Half width of absorption band at half height. reported are 95% confidence limits. acid chloride (0.011-0.013 mol) in 40 mL of tetrahydrofuran was added in a dropwise manner, The reaction mixture,was heated at approximately 50 “C for 3 h and maintained at pH 8 dr above. The reaction mixture was cooled and extracted with chloroform. The chloroform fraction was dried, evaporated, ahd theresulting residues purified by recrystallization using petroleum ether and benzene. Molar absorptivities for these comDounds were determined in the usual manner. Chromatographic Procedures. T6e liquid chromatograph was a modular isocratic system consisting of a Waters (Milford, Ma) 6000A pump, U6K injector, and 440 absorbance detector. Normal phase separation was achieved with either a 15.0 cm by 4.6 mm i.d. column packed with 5-pm porous Zorbax-Si1(Du Pont, Wilmington, DE) or Supelcosil LC-Si (Supelco, Inc., Bellefonte, PA). These columns were preceded by a 7.0 cm by 2.1 mm i.d. guard column which was dry packed with 30-38 wm HC Pellosil (Whatman, Clifton, NJ). The mobile phase consisted of various mixtures of HPLC grade chloroform and n-heptane at a flow rate of 1.4 mL/min. The UV detector was operated at 254 nm and 0.01 AUFS. The separations were carried out at ambient temperature and the column void volume was determined with toluene. Reversed-phase separation was accomplished by use of a 15.0 cm by 4.6 mm i.d. column packed with CI8 chemically bonded silica (5 Fm), Zorbax ODS (Du Pont, Wilmington, DE). The mobile phase consisted of mixtures of water and HPLC grade methanol, tetrahydrofuran, or acetonitrile. The flow rate was 1.5 mL/min and the UV detector was operated at 254 nm and 0.01 AUFS. These separations were carried out at ambient temperature and the column void volume was determined by using sodium nitrate (16) with background electrolyte.
nm
E mC
246 91 349 183 1560 860 163 259
14.0 14.0 11.0 11.0 34.0 34.0
Values
Scheme I
LLLOH+ I
X ~ S O 2 C I
H
‘OH
SOCI,
X
eSO 2 -N
g
c”p I ‘CI
I
so2
I
X X =H,OCH,, NOZgCI
RESULTS AND DISCUSSION Precolumn derivatization with a suitable resolving agent leads to the formation of diastereomers by the introduction of an additional chiral center in the molecule. These iliastereomers are then separable due to differences in their chemical and physical properties. Additionally, derivatization can function to increase the detectability of the products if the derivatizing agent contains highly detectable functional groups (chromophores for UV detection). The preparation of a chiral derivatizing agent having good chromophoric properties was the initial goal of this work. This was accomplished by converting the amino acid S-proline (L-(-)-proline) to various para-substituted phenylsulfonamides. Preliminary studies showed these sulfonamides to have good UV absorption properties. A set of model diastereomers were prepared by reacting the individual R and S isomers of a-methylbenzylamine with the corresponding acid chlorides of the para-substituted (phenylsulfony1)prolines (Scheme I). The physical properties of the eight diastereomers prepared in this
manner me presented in Table I. The UV absorption spectra of these compounds were evaluated as a measure of HPLC detectability. The wavelength of maximum absorption (A,=) and the molar absorptivity (e) at A,, and a t 254 nm (fixed wavelength commonly used in HPLC detectors) are reported in Table I. The (phenylsulfony1)prolylamide(R = H) showed above 200 nm and only weak absorption a t 254 nm. no A,, The other diastereomers showed strong absorbances at ,A, and the methoxy and nitro substituents (R = OCH, and NOz) displayed strong absorbances at 254 nm. The methoxyamide displayed the strongest absorbance a t Am;, however, the nitroamide had the broadest absorption band (Q)and the highest e value a t 254 nm. The properties of the model diastereomers in normal-phase and reversed-phase chromatography were examined as a measure of the ability of the chiral derivatizing agents to resolve amine enantiomers. The chromatographic parameters evaluated were capacity factors (k’), selectivity (a), and the
960
ANALYTICAL CHEMISTRY, VOL. 56, NO. 6, MAY 1984
Table 11. Normal-Phase Chromatographic Data for Model Diastereomers at Various Solvent Strengthsa
O
90% CHCl, 10% heDtane
100% CHCl,
X H H
configuration
k'
1.21 1.61 c1 0.83 c1 1.20 OCH, 1.43 OCH, 1.90 NO2 1.43 NO2 1.82 a Column: Zorbax-Sil.
CY
Rs
S ,R
s,s S,R s,s S, R s,s S,R s,s
H
1.33
0.93
1.44
0.77
1.33
1.44
1.27
1.00
k'
1.72 2.30 1.20 1.70 2.06 2.67 2.20 2.79
resolution (R,) of the diastereomeric pair. The normal phase data are presented in Table 11. Most reported separations of amide diastereomers have used normal-phase systems (IO, 17). Each of the four sets of diastereomers were separated in a mobile phase of pure chloroform. The separation improved upon addition of heptane to the mobile phase and R, > 1was achieved for each diastereomeric pair at 10% (v/v) heptane in chloroform. The adsorption energy of a molecule should equal the sum of the adsorption energies of the individual constituent groups (18). These diastereomers differ in substituent groups only at the para position of the phenylsulfonyl group and the observed elution order parallels the adsorption energies (Qivalues) for these groups (19),Qi for C1< H < OCH3 < NOz. Comparison of the It' values for the four compounds of each diastereomeric form (S,S or S,R) shows this same elution order. Examination of the elution characteristics of each individual set of diastereomers shows the S,S diastereomer to have a higher adsorption energy than the S,R form in each of the four cases examined. The reversed-phase chromatographic properties of these diastereomers were examined by using a hydrocarbonaceous (CIS)stationary phase and binary solvent mixtures of water with methanol, acetonitrile, and tetrahydrofuran. The only diastereomers separated were the 1-[ (4-nitropheny1)sulfonyl]prolines and this separation was achieved only in a water-methanol mobile phase (Figure 1). Matsutera et al. (20) have suggested that the reversed-phase resolution of diastereomersrequires a mobile phase organic modifier having either hydroxyl or ether groups. Furthermore only the electron-withdrawing nitro group produced reversed-phase separation and the elution order was S,S before S,R, the reverse of the normal-phase elution pattern. These results suggested that 1-[(4-nitrophenyl)sulfonyl]prolyl chloride (NPSP-C1)was the most versatile of the chiral derivatizing agents for the separation of mixtures of amine enantiomers. This chiral reagent imparted strong UV absorption properties to the resulting diastereomers as well as a broad absorption band which included 254 nm. Separations were achieved in both normal-phase and reversed-phase chromatographic systems. Additionally, NPSP-C1 is a relatively stable crystalline solid easily purified by recrystallization. No significant racemization was observed during the derivatization process with any of the chiral reagents. The utility of NPSP-Cl for the determination of the enantiomeric composition of other primary and secondary amines was confirmed by the resolution of amphetamine and the ephedrines. Amphetamine contains a single chiral center and the resulting two stereoisomers are mirror images of each other (enantiomeric). The ephedrine molecules, however,
80% CHCl, hentane _20% _ .. .. r r - - - -
RS
CY
k'
1.34
1.24
1.42
1.50
1.34
1.60
1.27
1.45
CY
2.28 3.13 1.58 2.32 2.83 3.94 3.17 4.09
o=s=o
RS
1.38
1.65
1.47
2.35
1.39
2.11
1.29
1.89
1
1
=s
2- R
0
2
4
8
8 1 0
T i m e (minutes)
separation of the NPSP derivatives of a-methylbenzylamine in methanol-water (6 + 4).
Figure 1. RPLC
contain two chiral centers resulting in four possible stereoisomers, two sets of enantiomers. The erythro isomers (R,S and S,R) are known as ephedrine and the threo isomers (R,R and S,S) referred to as pseudoephedrine. The relationship between the erythro and threo forms is diastereomeric, one chiral center of the same configuration and one of opposite configuration. Derivatization of racemic amphetamine with NPSP-C1 yields the diastereomeric NPSP-amides (S,R and S,S). The liquid chromatographic separation of the amphetamine-NPSP derivatives on an achiral silica stationary phase is shown in Figure 2. The diastereomers are easily resolved in a chloroform-heptane solvent system requiring approximately 5 min. However, these diastereomers are not readily resolved by reversed-phase techniques. The failure of the amphetamine-NPSP derivatives to separate under reversed-phase conditions appears to be an anomaly. This is supported by the chromatographic properties of the NPSP derivatives of a-ethylbenzylamine. A sample of a-ethylbenzylamine (1amino-1-phenylpropane),a positional isomer of amphetamine, was synthesized, derivatized with NPSP-Cl, and easily sepa-
ANALYTICAL CHEMISTRY, VOL. 56, NO. 6, MAY 1984
961
a, b
E
1 -R,S
2 1 S.R 3.S.S 4-R,R
3hl Y
Q) v)
C
1=R 2= s
g v)
8 L
0
C
0 Q) C
8
0
2
3
4
5
6
7
8
9
Time (minutes) 0
1
2
3
4
Flgure 4. RPLC separation of the ephedrine- and pseudoephedrineNPSP derivatives in methanol-water (6 4).
+
5
Time (minutes) Figure 2. Normal-phase separation of amphetamine-NPSP derivatives 2). on Supelcosil LC-Si in chloroform-heptane (8
+
a , b l = R . S 2 = S , R
Time (minutes) Figure 3. Normal-phase separation of ephedrine-NPSP derivatives on 1). Supelcosii LC-Si in chloroform-heptane (9
+
rated in a reversed-phase system. Since the NPSP moiety is the same in both sets of diastereomers the difference in chromatographic properties must lie with the amine portion of the molecule. Oki and Mutai (21) have reported intrarholecular hydrogen bonds of the N-H--?r type in N-benzylanilines and N-phenethylanilines. Such a six-membered intramolecular hydrogen bond may play a role in the inhibition of the reversed-phase resolution of the amphetamine-NPSP derivatives. The normal phase separation of the diastereomeric ephedrine-NPSP derivatives (S,R,S and S,S,R) is shown in Figure 3. The pseudoephedrine-NPSP derivatives were also
separable under the same conditions; however, complete resolution of all four isomers was not achieved by normal phase. The compounds were also separable by reversed-phase liquid chromatography in a methanol-water solvent system. Figure 4 shows the resolution of all four diastereomers: peak 1 and 2 are the ephedrine-NPSP derivatives and peaks 3 and 4 are the pseudoephedrine-NPSP derivatives. The elution order of the two ephedrine-NPSP derivatives on the reversed-phase column is opposite to that in normal phase. The same elution order reversal was observed for the pseudoephedrine-NPSP derivatives. However, the two ephedrineNPSP derivatives eluted before the two pseudoephedrineNPSP derivatives in both normal-phase and reversed-phase systems. In principle the employment of chiral derivatizing agents to achieve indirect resolution of amines is less desirable than direct chromatographic resolution. However, for primary amines, direct methods have not evolved to the point where they are competitive in scope or scale with indirect methods (22). NPSP-C1 appears to be a very useful chiral derivatizing agent for the conversion of enantiomeric primary and secondary amines to diastereomeric products. These diastereomers are subject to liquid chromatographic separation on standard achiral stationary phases using either normal-phase or reversed-phase techniques. Registry No. (S,R)-PhS02ProNHCH(CH3)Ph,88867-88-3; ( S , S )-PhS 0 2 P roNHCH ( CH,) P h, 88867 - 89-4; ( S , R) -4C1C6H4S02ProNHCH(CH3)Ph, 88867-90-7; (S,S)-4C1C6H4SO2ProNHCH(CH3)Ph, 88867-91-8; (S,R)-4MeOC6H4S02ProNHCH(CH3)Ph, 88867-92-9; (S,S)-4MeOC6H4S02ProNHCH(CH3)Ph, 88867-93-0; (S,R)-4O2NCBH4SO2ProNHCH(CH3)Ph,88867-94-1; (S,S)-402NC6H4S02ProNHCH(CH3)Ph, 88867-95-2;PhSO2C1,98-09-9; 4-CIC6H4S02Cl, 98-60-2; 4-MeOC6H4S02CI, 98-68-0; 402NC6H4S02C1,98-74-8; L-PhS02ProOH, 88425-46-1; L-4C1C6H4S02ProOH, 73096-27-2;L-4-MeOC6H4S02ProOH, 8124227-5; L-~-O,NC~H,SO~P~OOH, 88867-96-3; (S)-PhS02ProCl, 72922-83-9; (S)-4-ClC6H4S02PrOC1, 88867-97-4; (S)-4MeOC,H4S02ProCl, 88867-98-5; NPSP-Cl, 88867-99-6; (S,R)amphetamine-NPSP, 88868-00-2; (S,S)-amphetamine-NPSP, 88868-01-3; (S,R)-a-ethylbenzylamine-NPSP, 88868-02-4; (S,SI-a-ethylbenzylamine-NPSP, 88868-04-6; (S,R,S)-ephedrineNPSP, 88868-03-5; (S,S,R)-ephedrine-NPSP,88928-95-4; (S,S,S)-pseudoephedrine-NPSP, 88928-96-5; (S,R,R)-pseudoephedrine-NPSP,88928-97-6;(*)-a-methylbenzylamine, 618-36-0;
962
Anal. Chem. 1984, 56,962-966
(R)-a-methylbenzylamine, 3886-69-9; (S)-a-methylbenzylamine, 2627-86-3; amphetamine, 300-62-9; (*)-ephedrine, 90-81-3; (*)-a-ethylbenzylamine, 35600-74-9; L-proline, 147-85-3; (*)pseudoephedrine, 4125-58-0.
LITERATURE CITED Kotake, M.; Sakan, T.; Nakamura, N.; Senoh, S. J . Am. Chem. SOC. 1951, 73, 2973. Gii-Av, E.; Nurok, D. “Advances in Chromatography”; Giddings, J., Keiier, R.,Eds.; Marcel Dekker: New York, 1974; Voi. 10, pp 99-172. Lochmuiier, C.,;Souter, R. J . Chromatogr. 1975, 113,283. Hara, S.;Dobashi, A. J . Liq. Chromafogr. 1979, 2 , 883. Baczuk, R.; Landram, G.; Dubois, R.; Dehm, H. 2. Chromatogr. 1971, 60,351. Dotsevi, G.; Sogah, Y.; Cram, D. J . Am. Chem. SOC. 1975, 97, 1259.
Hesse, G.; Hagei, R. Chromafographia 1973, 6 ,277. Heimchen, G.; Niii, G.; Fiockerzi, D.; Schuhie, W.; Youssef, M. Angew. Chem., Inf. Ed. Engl. 1979, 18, 62. Pirkie, W.; Hauske, J. J . Org. Chem. 1977, 42, 1839. Stoii, A.; Hofmann, A. Z . Physioi. Chem. 1938, 151,155.
(11) Tamegai, T.; Ohmae, M.; Kawabe, K.; Tomoeda, M. J . Liq. Chromafogr. 1979, 2, 1229. Low, G.; Haddad, P.; Duffieid, A. J . Liq. Chromatogr. 1983, 6 ,311. Liu, J.; Ramesh, S; Tsay. J.; Ku, W.; Fitzgeraid, M.; Angeios, S.; Lons, C. J . Forensic Sci. 1981, 26, 656. Liu, J. J . Forensic Sci. 1981,26,651. Hufsey, J.; Copper, D. Microgram 1979, 12,231. Wells, M.; Clark, R. Anal. Chem. 1981, 53, 1341. Lindner, W. Chimica 1981, 35, 294. Snyder, L “Chromatography: A Laboratory Handbook of Chromatographic and Electrophoretic Methods”, 3rd ed.; Heftmann, E., Ed.; Van Nostrand Reinhold: New York, 1975; pp 46-75. Snyder, L. “Principlesof Adsorption Chromatography”; Marcel Dekker: New York, 1968; pp 23-52. Matsutera, E.; Nobuhara, Y.; Nakanishi, Y. J . Chromafogr. 1981, 216, 374. Oki, M.; Matai, K. Bull. Chem. SOC.Jpn. 1965, 38, 387. Pirkie, W.; Simmons, K. J . Org. Chem. 1983, 48, 2520.
~~~~
RECEIVED for review August 16,1983. Accepted January 16, 1984.
Preconcentration and Multicomponent Chromatographic Determination of Biological Carbonyl Compounds James Raymer, Mary L. Holland,’ Donald P. Wiesler, and Milos Novotny*
Department of Chemistry, Indiana University, Bloomington, Indiana 47405
A method for the Isolation and analytical separatlon of aldehydes and ketones from blologlcal media has been developed. The methodology employs an Ion-exchange removal of acldlc and basic constituents followed by concentratlon of the remaining neutrals onto a modified adsorbent. The neutrals are subsequently eluted wlth methanol, and derlvatlzed wlth 2,4-( dlnltropheny1)hydrazlne under acidic condltlons. The resulting derivatives are purified and subjected to reversedphase HPLC with a water/acetonitrlle gradient. The method Is linear over a wlde concentratlon range and low nanogram detectlon llmlts are demonstrated for standard compounds. Two appllcatlons of this methodology to blologlcal problems are briefly descrlbed.
Carbonyl compounds occur widely in various samples of biological and environmental interest. During recent years, many investigations have been directed toward the development of methodology suitable for the analysis of aldehydes and ketones at low concentrations; studies in this area were recently reviewed (1). As these carbonyl compounds are frequently encountered in complex sample matrices, efficient chromatographic methods have been increasingly popular. While volatile carbonyl substances can be analyzed through gas chromatography (GC), many newer procedures tend to favor high-performance liquid chromatography (HPLC) to cover a wide range of aldehydes and ketones in various samples. Moreover, to facilitate detection in HPLC, some sort of derivatization is needed. Formation of dinitrophenylhydrazones is among the most popular approaches to the analysis of aldehydes and ketones (2). This derivatization principle has been incorporated into many procedures, including the analysis of formaldehyde and Present address: MacNeil Laboratories, Spring House, PA 19477.
related compounds in automobile exhaust (2) and tobacco smoke (3),both aliphatic and aromatic aldehydes in polluted air ( 4 ) ,and nanogram quantities of various carbonyls in air samples (5). In addition, aqueous samples were analyzed for similar substances (6). Selim ( 7 ) reported the addition of isooctane to an aqueous reaction mixture in order to extract the derivative after its formation. It is assumed that this modification yields equilibrium conditions more favorable to product formation in the aqueous layer, thus giving rise to a more complete derivatization. Thus far, little attention has been paid to the analysis of carbonyl compounds in biological media where the concentrations of endogenous aldehydes and ketones are low. Specific inquiries in our laboratory, in this respect, have concerned the role of certain carbonyl compounds in human metabolism (8) and as potential chemical messengers of mammals (9). In both cases, it became necessary to give special attention to (a) efficient preconcentration (from 50 to 500 ng/mL initial concentrations) prior to analysis, (b) removal of interfering substances (e.g., keto acids whose concentrations can be substantial), and (c) an effective separation of (possibly) related metabolites, followed by a quantitative measurement of the individual carbonyls. Simultaneously, the steps needed to accomplish these goals should be free, as much as possible, from interferences originating in solvents, reagents, etc., as greatly reduced sample volumes are necessarily involved. Accordingly, an effective procedure for isolation of carbonyl compounds and sample preconcentration, followed by derivatization and HPLC-based measurements, has been developed here. Quantitative aspects are described, as are two biologically significant illustrations of this procedure, which should prove to be of general application.
EXPERIMENTAL SECTION DEAE-Sephadex anion exchanger (Sigma Chemical, St. Louis, MO) was equilibrated in 0.05 M Tris buffer at pH 8.3 with 0.02% sodium azide as a preservative. CM-Sephadex cation exchanger
0 1984 American Chemical Society 0003-2700/84/0356-0962$01.50/0
