Anal. Chem. 1900, 60, 2131-2134
CONCLUSIONS The aqueous phase modifier y-CDx has been studied for its utility in separating PAH mixtures by solvent extraction. Preliminary results suggest that this technique has potential use for separating PAHs on the basis of molecular dimensions. If specific modifications are made, the possibility of separating mixtures may be enhanced. The best use for this system may be as a method for simplifying complex mixtures by partitioning groups of compounds between the organic and aqueous phases. Many applications for CDx-modified extraction may be found in the motor industry, in air sampling analysis, and in many types of industrial products that contain PAHs as primary components. Also, combining the extraction technique described here with data analysis techniques such as pattern recognition may allow for complete identification of all species present in complex mixtures. LITERATURE CITED Szejtii. J. Cyclodextrins and Their Inclusion Complexes ; Akademiai Kiado: Budapest, 1982. Saenger, W. Angew. Chem., Int. Ed. Engl. 1980, 19, 344-362. Juks, 0.; Scypinski, S.; Cline-Love, L. J. Anal. Chim. Acta 1985, 169, 355-360. ... ... Scypinski, S.; Cline-Love, L. J. Anal. Chem. 1984, 5 6 , 322-327.
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Cline-Love, L. J.; Grayeski, M. L.; Noroski, J.; Weinberger, R. Anal. Chim. Acta 1985, 170, 3-12. Breslow, R.; Kohn, U.; Siegel, B. Tetrahedron Left. 1978. 1645-1646. Beesley. T. E. Am. Lab. (FaitfieM, Conn.) 1985, May, 78-87. Smolkova-Keulemansova, E. J . Chromatogr . 1982, 25 1 , 17-34. Natusch, D. F. S.; Tomkins, B. A. Anal. Chem. 1978, 50, 1429-1434. Matsunaga, K.; Imanaka, M.; Ishlda, T.; Oda, T. Anal. Chem. 1984, 56, 1980-1982. Harangi, J.; Nanasi, P. Anal. Chim. Acta 1984, 156, 103-109. Szejtli, J. German Patent 2927733, 1980; Chem. Abstr. 1980, 9 2 , 181902. Nakai, Y.; Yamamoto, K.; Terada, K.; Horibe, H. Chem. Pharm. Bull. 1982, 30(5), 1796-1802. Street, K. W. J. Liq. Chromatogr. 1987, 10(4), 655-662. Kalyanasundaram, K.; Thomas, J. K. J. Am. Chem. SOC. 1977, 9 9 , 2039-2044. Singh, H.; Hinze, W. L. Analyst(London)1982, 107, 1073-1080. de Moreno, M. R.; Smith, R . V. Anal. Lett. 1983, 16(A20), 1633-1 .. - - .645. .... Hamai, S. Bull. Chem. SOC.Jpn. 1982, 5 5 , 2721-2729. Warner, I. M.; Fogarty, M. P.; Shelly, D. C. Anal. Chim. Acta 1979, 109. 361-372.
RECEIVED for review March 14,1988. Accepted May 24, 1988. This work was supported in part by the National Science Foundation (CHE-8609372) and the Office of Naval Research. Isiah M. Warner acknowledges support from an NSF Presidential Young Investigator Award (CHE-8351675).
Separation of Deuteriated Isotopomers of Dopamine by Ion-Pair Reversed-Phase High-Performance Liquid Chromatography Carolyn F. Masters, Sanford P. Markey, and Ivan N. Mefford* Laboratory of Clinical Science, National Institute of Mental Health, Building 10, Room 2046, 9000 Rockville Pike, Bethesda, Maryland 20892
Mark W. Duncan Intramural Research Program, National Institute of Neurological and Communicative Disorders and Stroke, 9000 Rockville Pike, Bethesda; Maryland 20892
The ion-pair reversed-phase separation of dopamine and deuterium-substituted dopamine isotopomers is described. Chromatographlc parameters and deuterium isotope effects governlng the resolutlon are examined and compared to the factors reguiatlng the resolution of the chemically distinct entities dopamine, noreplnephrine, and epinephrine. The potential utility of the ['H,]dopamine isotopomer as an internal standard for the high-performance liquid chromatography analysis of dopamine is demonstrated by using aluminum oxide extraction prior to chromatographic Separation.
Stable isotope analogues have been extensively employed in the biological sciences. Quantitative methodologies based on mass spectrometric analysis have taken advantage of the fact that isotopomers are ideal mimics for the chemical behavior of a compound, better than either homologues or structural analogues, and the isotopomers can be distinguished and independently quantified. However, analysis methods incapable of mass discrimination require chromatographic separation for general application of stable isotopes. Whereas
* A u t h o r t o w h o m correspondence should b e addressed.
separation of deuterium-substituted compounds from their protium isotopomers has been possible by using gas chromatographic techniques for many years ( 1 , 2 ) , liquid chromatographic approaches have not commonly been used for this purpose. With the advent of high-resolution columns packed with hydrophobic stationary phases, it has been demonstrated that such separations are possible (3-6). The successful liquid chromatographic separation of deuteriated isotopomers makes possible their routine use as internal standards without introducing the need for mass specific detection or the application of radiolabeled isotopes. The ability to resolve deuteriated isotopomers of neurochemically active analogues would also make it possible to perform tracer and turnover studies in animals and humans without the safety hazard associated with the use of radioactive isotopes. Previously published separations of biochemically relevant isotopomers have generally not been useful for routine analyses for several reasons. Near complete resolution has been accomplished only for fully deuteriated molecules, some with more than 30 hydrogens exchanged (3-6). Separations of partially deuteriated compounds have only been accomplished on very long microbore columns (4.5 m X 1 mm i.d.) (6). These separations require exceedingly long analysis times as the flow rate must be kept low in order to maintain acceptable column back pressure (3, 4 , 6). Practical separation of carotenoid
This article not subject to U S . Copyright. Published 1988 by the American Chemical Society
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isotopomers has however been reported (5). These seminal works have indicated that such separations using high-performance liquid chromatography (HPLC) techniques are possible. The chromatographic separation of catecholamines by ion-pair reversed-phase HPLC has been extensively investigated over the past decade and is now routinely performed. Literally hundreds of different separation schemes using a variety of reversed-phase columns and ion-pair reagents have been published (see ref 7 for a review of these applications). These separations have used either 3,4-dihydroxybenzylamine or a substituted dopamine analogue, either N-methyldopamine (8) or a-methyldopamine (9) as the internal standard. These internal standards differ in their reactivity when compared to the catecholamines being quantified. Unlike dopamine or the other catecholamines of interest, autooxidation of 3,4dihydroxybenzylamine cannot lead to an intramolecular cyclization product (10). The N-methyldopamine analogue can cyclize more readily than dopamine, as is the case for epinephrine compared to norepinephrine (11) and could be formed in vivo. The a-methyldopamine analogue increases analysis time for catecholamines as it is retained significantly longer than dopamine. Finally, all these catecholamine analogues have modestly different extraction efficiencies through either the classical aluminum oxide procedure or diphenylborate (12)than dopamine, making these compounds less than ideal internal standards. The separation of catecholamine isotopomers by HPLC has not been reported, although species with 2H, to 2H7are routinely used in gas chromatography/mass spectrometry (GC/MS) applications. Our interest was to explore the possible application of deuteriated isotopomers of dopamine as potential internal standards for the analysis of dopamine, as well as the possible utility of deuteriated precursors of dopamine as tracers for metabolic studies eliminating the hazards associated with the use of radioactive isotopes. The first requirement for further application of these compounds was chromatographic resolution. This paper examines approaches to the chromatographic resolution of deuteriated dopamine isotopomers and discusses the different mechanisms involved in the separation of catecholamines by ion-pair reversed-phase HPLC. EXPERIMENTAL SECTION Deuteriated Dopamine Analogues. 2-(3,4-Dihydroxypheny1)ethyl-l,l,2,2-d4-aminehydrochloride ( [2H4]dopamine)and 2-(3,4-dihydroxyphenyl)ethyl-l,l-d2-amine hydrochloride ( [2H2]dopamine)were obtained commercially from Merck Isotopes (St. Louis, MO). [2H7]Dopaminewas prepared from ['H4]dopamine by exchange according to the procedure of Vining et al. (13). Gas chromatography/mass spectrometry of the pentafluoropropionyl (PFP) derivative of the exchange product was used to establish the chemical and isotopic purity of the product. The derivatized product was shown to consistent of only one component, which cochromatographed with the PFP derivative of authentic dopamine. The base peak in the E1 spectrum of a scan from this GC peak was shown to be increased by 3 mass units (from m / z 431 to 434) when compared with the [2H4]dopamine used as starting material. The absence of other ions in the range m / z 428-434 established that the product was predominantly the [2H,]isotopomer. Chromatography. Liquid chromatographic separation was performed on a 7.5 cm length X 4.6 mm i.d. ODS column packed with 3-pm Ultrasphere (Beckman). Mobile phases were prepared with varying concentrations of NaH2P04in order to study the influence of ionic strength on chromatographic resolution. Mobile phase polarity was modified with methanol in order to study the effects of organic solvents on the resolution of dopamine analogues. Ion pairing concentration in the mobile phase was held constant, using sodium dodecyl sulfate (Kodak) at 100 mg/L. pH effects were not studied as both catecholamines and the alkyl sulfate remain charged in the pH range of silica-based reversed-phase
A
NUMBER OF DEUTERONS
B
NUMBER OF DEUTERONS
Flgure 1. Effect of deuterium substitution on capacity factor (A) and resolution of deuteriated and protium isotopomers of dopamine (E). HPLC column was 7.5 cm X 4.6 mm i.d., packed with 3-pm ODs Ultrasphere (Beckman). Solvent conditions were as follows: 0.7 M NaH,PO,, 100 mg/L SDS, 0% methanol; flow rate, ~ 1 . mL/min. 0
materials, pH 2-7. Solutions used as mobile phase were filtered through 0.45-pm fiiters under vacuum and degassed prior to use. Instrumentation. Pulseless solvent delivery was accomplished with a Model 400-02 reciprocating pump (Applied Chromatog raphy Systems, Inc.). Sample injection (50-100 pL) was accomplished with a fixed 100 /IL loop injector (Gilson Autosampler, Model 231). Chromatograms were recorded on a strip chart recorder (LKB Instruments). Catecholamines were detected amperometrically by using a glassy carbon electrode (TL-8A) at a potential of +0.6 V vs Ag/AgCl reference with a Model LC-4B amperometric detector (Bioanalytical Systems, Inc.). Amperometric cell volume was minimized by use of a Wpm Teflon gasket. Extraction of Catecholamines. A batch procedure was used to extract catecholamines. Quantities (100 pL of 1.0 X 10" M) of catecholamines, norepinephrine, epinephrine, 3,4-dihydroxybenzylamine, [2H7]dopamine,and [2Ho]dopaminewere aliquoted into a 1.5-mL polypropylene tube. To this was added 20 mg of acid-washedaluminum oxide (Woehlm, 200 mesh), and the volume brought to 1.5 mL with 1.5 M Tris HCI, pH 8.6. Tubes were shaken for 15 min and the alumina was washed twice with distilled deionized water. Alumina was aspirated to dryness and the catecholamines desorbed with 100 pL of 1% acetic acid (v/v). Recoveries of the catecholamines were compared to 3,4-dihydroxybenzylamine and [2H7]dopamine.
RESULTS AND DISCUSSION Effects of Deuterium Substitution. The effects of the degree of deuterium substitution on capacity factor ( k ? and resolution (R)were studied. These data are shown in Figure 1. Both capacity factor and resolution increased with increasing deuterium substitution. The effect of additional deuterium substitution was not linear, thus the isotope effect was smaller with each additional substitution. Depending upon the number of substituted deuterons, the average isotope effect was 1.34% (2H7), 1.87% (2H4),and 1.68% (2H2)of k' per deuterium. Deuterium substitution on the side chain (2H4 and 2H.J had a relatively greater effect on k 'than substitution on the benzene ring. Maximum resolution ( R = 1.2) was obtained with the maximally substituted analogue, 2H7(Figure 2). Chromatographic tracings showing the degree of separation between the deuterium isotopomers of dopamine are shown in Figure 2. Some distinction in isotope effect deter-
ANALYTICAL CHEMISTRY, VOL. 60, NO. 19, OCTOBER 1, 1988
2H7 II .
I
Ra
JUL
Flgure 2. Chromatograms demonstratingthe separations achieved for the various isotopomers of dopamine. Chromatographic condiins are given in Figure 1.
mined by position of substitution might be found in these data, as the 2Hzand 2H4analogues are side chain substituted while the three additional deuteriums found in the 2H7analogue are incorporated in the benzene ring. Thus, isotope effects caused by the deuterium atoms attached to the ethylamine side chain might be greater than that associated with substitution onto the benzene ring. Nearly base-line resolution ( R = 1.2) was obtained between the [2Ho]-and [2H7]dopamineisotopomers. The resolution of 2Ho-and 2H7-substituteddopamines is accomplished with a commercial, conventional bore column providing only 8700 theoretical plates. At least substitution would appear to be necessary to achieve sufficient resolution (Le. R > 0.8) for tracer studies. The use of [2H7]tyrosineas the precursor for studies of catecholamine metabolism should allow the biosynthesis of [2H6]dopamine and analogues of norepinephrine, epinephrine, and metabolites of dopamine, 3,4dihydroxyphenylacetic acid, and homovanillic acid. This should provide sufficient resolution for studies of turnover, synthesis, and degradation. Effects of Mobile Phase Parameters. The effects of mobile phase parameters were studied to determine which factors could be manipulated to modify resolution of the various analogues. Figure 3 demonstrates the effect of manipulation of ionic strength through altering the phosphate concentration on k'of ['H]catecholamines and [2H7]dopamine. The dopamine isotopomers behave almost identically, Figure 3A, while norepinephrine and epinephrine are differentially affected, Figure 3B. This is demonstrated more effectively by considering the effect of ionic strength on resolution. The
k
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0 1 . ' " " . 0.0 0.1 0 . 2
'
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.
'
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'
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2133
resolution of [2Ho]-from [2H7]dopamineis unaffected by ionic strength. By contrast, the resolution of norepinephrine from epinephrine, which differs only in methyl substitution on the charged nitrogen, is affected. These data demonstrate that the mechanism that chromatographically differentiates isotopomers differs from that involved in the resolution of chemically distinct entities such as norepinephrine and epinephrine. Epinephrine and norepinephrine are differentiated partially through ionic interactions presumably due to the order of substitution on the ethanolamine nitrogen. By contrast, the deuteriated dopamine isotopomers are i d e n t i d y affected through the ionic portion of the molecule. We did observe that at low ionic strength, which produced very long retention times, the resolution between the dopamine isotopomers was slightly lower, R = 0.9. This is likely due to increased dispersion due to diffusion during the long elution time, a problem encountered by others in the resolution of 2H and 'H isotopomers by conventional (macro) bore reversed-phase HPLC. In contrast, epinephrine, norepinephrine, and 2Ho are all similarly affected by the addition of organic modifier to the mobile phase (see Figure 4). Both resolution and capacity factors are affected similarly between norepinephrine and epinephrine as well as [2H7]-and ['HIdopamine, decreasing with increasing organic modifier in the mobile phase (see Figure 4). This indicates that the effect of mobile phase polarity modification is general, modifying the relative solubility of the analytes in the mobile and stationary phases and is likely related to similar properties of these compounds. The deuteriated dopamine isotopomers eluted first under all chromatographic conditions used. This is consistent with the observations of Baweja (5) who achieved base-line resolution of carotenoids, also using an Ultrasphere ODS column. Deuteriated isotopomers of both lutein and @-carotenemoved more rapidly through the column than did the protio analogues. In these cases, the carotenoids were fully deuteriated, containing 54 and 56 deuteriums, respectively. We have achieved nearly base-line separation of dopamine isotopomers with only seven deuterium substitutions. The column efficiency was slightly higher in the present study, N i= 8000 (100000 plates/m), while N i= 6500 (26000 plates/m) for the separation of lutein shown by Baweja (5). [ N was calculated as N = 5.54(t,/t,1/2)2.] Separation of deuteriated compounds by normal-phase chromatography has been demonstrated for caratenoids on magnesia columns (14). The order of elution was reversed to that on reverse phase, with the deuteriumsubstituted isotopomers eluting more slowly. We attempted unsuccessfully to separate deuteriated isotopomers of catecholamines by cation exchange liquid chromatography several years ago (Adams, R. N.; Mefford, I. N.; Keller, R. W., Jr., unpublished observations). Although the chromatographic efficiency in the present work is significantly better, this 300
-
200
-
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0.0
0 . 1
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0.5
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Figure 3. Effect of mobile phase salt concentratbn on capacity factors of norepinephrineand epinephrine (A) and [2H,]dopamine and [21-b]dopamine (B). HPLC column Is given in Figure 1. Salt concentration was varied while other moblle phase conditions were held constant.
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ANALYTICAL CHEMISTRY, VOL. 60, NO. 19,
'.* 1
OCTOBER 1, 1988
Table I. Recovery of Catecholamines Relative to Dihydroxybenzylamine and [2H,]Dopamine4
A
vs dihydroxybenzylamine
dopamine epinephrine norepinephrine
105.4 f 0.6% 101.7 f 2.2% 100.5 f 1.5%
vs [*H7]dopamine 99.7 f 0.5% 96.2 f 2.3% 95.1 f 1.6%
These values represent the means of nine determinations f the relative standard deviation.
-
0.2
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DA 1
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0
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B
j
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20
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Flgure 4. Relative effect of varying solvent methanol content on capacity factors of catecholamines (A) and resolution of norepinephrine vs epinephrine and [2H,]dopamine vs ['H,]dopamine (B). Salt and SDS concentrations were held constant while the methanol content was varied. All values were normalized to k'(A) or R (B) at 0% methanol. Column type is given in Figure 1.
observation also supports the lack of involvement of the protonated amine in distinguishing these compounds. The only alkyl sulfate/sulfonate tested for which data have been presented was sodium dodecyl sulfate. We did obtain similar resolution of the deuteriated dopamine isotopomers by using a novel cation exchange reagent, N-methyloleoyl taurate (10). Many similar separations of catecholamines by ion-pair reversed-phase HPLC employ shorter chain alkyl sulfates and alkylsulfonates as the ion pair reagents. It is possible that these shorter chain anions may provide somewhat different selectivities than observed here and may increase the reversed-phase character of the separation. Extraction of Catecholamines. A second object of these experiments was to demonstrate that deuterium-substituted isotopomers of dopamine would be suitable internal standards for the determination of catecholamines. By comparison of the extraction of [2H7]-and ['Hldopamine with the commonly used internal standard for catecholamine extraction and determination, 3,4-dihydroxybenzylamine,it was possible to determine the suitability of the deuteriated isotopomer for this purpose. The relative recoveries of these compounds taken through the aluminum oxide extraction are shown in Table I. The recoveries of the dopamine isotopomers through this procedure are essentially identical while consistent differences exist for the three ['H]catecholamines when compared to 3,4-dihydroxybenzy1amineaFixed differences also exist between deuteriated dopamine and the catecholamines norepinephrine and epinephrine. In fact, these data would indicate that [2H7]dopamineis less suited as an internal standard for these two catecholamines than 3,4-dihydroxybenzylamine. There was no observable isotope effect on extraction efficiencies between the dopamine isotopomers. This is antici-
pated as the adsorption to alumina occws through the vicinal 3,4-dihydroxy groups which are protonated in both species. These data demonstrate that deuteriated isotopomers of catecholamines incorporating five or more deuterons may be suitable for use as internal standards in quantitative HPLC analysis. The present data confirm the observations of many others, that 3,4-dihydroxybenzylamineis a suitable internal standard for catecholamine determination by HPLC with electrochemical detection following aluminum oxide extraction, comparable to the deuteriated dopamine analogue. In the case of dopamine, separation of the deuteriated isotopomers does not appear to be related to the ion exchange properties of the column or mobile phase. Polarity of the solvent does affect separation, and maximum resolution can be obtained with the least organic modifier in the mobile phase. Deuteriated isotopomers elute more rapidly than the [ 'Hldopamine, and the k ' value decreases with increasing numbers of deuterium substitutions. The effect of side chain deuteriation appears to be slightly greater than ring deuteriation. The isotope effect on k'ranges between 1.3 and 1.87% per deuterium. Separation of dopamine isotopomers is obtained to R = 1.2 when all seven carbon-bonded hydrogens were replaced by deuterium atoms, a change of only 4.6% in molecular mass. Further, the separation is accomplished on a commercial reversed-phase column (7.5 cm in length X 4.6 mm i.d.) and with electrochemical detection. These data suggest that using presently available reversed-phase materials, it is possible to achieve quantitative resolution of stable isotopomers of catecholamines. It appears likely that similar degrees of deuterium exchange in small molecules might allow resolution and general application of this approach to quantitative analysis and tracer studies. Registry No. Dopamine, 51-61-6; epinephrine, 51-43-4; norepinephrine, 51-41-2; [2H2]dopamine,72535-00-3;[2H4]dopamine, 115797-26-7;[2H7]dopamine,115797-27-8. LITERATURE CITED (1) Bentley, R.; Saha, C.; Sweeley, C. C. Anal. Chem. 1985, 3 7 , 1118. (2) Wilzbach, K. E.; Reisz, P. Science (Washington, D . C . ) 1957, 726, 748. (3) Pratt, J. J. Ann. Clin. Biochem. 1988, 2 3 , 251. (4) Kucera, P.; Manius, G. J. Chromafogr. 1981, 2 1 6 , 9. (5) Baweja, R. J. Liq. Chromatogr. 1888, 9, 2609. (6) Tanaka, N.; Thornton, E. R. J. Am. Chem. SOC. 1976, 98 1617. (7) Hashimoto, H.; Maruyama. Y. Methods in Biogenic Amine Research; Parvez, S., Nagatsu, T., Nagatsu, I., Parvez, H., Eds.; Eisevler: Amsterdam, 1983; p 35. (8) Scheinin, M.; Seooala, T.; Kouiu. M.; Linnoila. M. Acta Pharmacal. Toxicol. 1984,55; 88. (9) Jonsson, G.;Hallman, H.; Mefford, I . ; Adams, R. Central Adrenaline Neurons: Fuxe. K.. Goldstein.. M.., Hokfelt.. B... Hokfelt., T... Eds.:. Pergammon; Oxford, '1980;p 59. (10) Mefford. I. N.; Ota, M.; Stipetic, M.; Singleton, W. J. Chromafogr. 1987, 420, 24 1. (11) Hawley, M. D.; Tatawawadi, S. V.; Piekarski, S.; Adams, R. N. J. Am. Chem. SOC.1967, 89, 447. (12) Macdonaid, 1. A,; Lake, D. M. J. Neurosci. Methods 1985, 73, 239. (13) Vlnina, R. F.: Smvthe, G.A.; Long. M. A. J. LabeliedCom~d.Radiopharm: 1981, 78; 1683. (14) Strain. H. H.; Thomas, M. R . Crespi, H. L.; Katz, J. J. Biochim. Biophys. Acta 1961, 5 2 , 517.
RECEIVED for review February 25, 1988. Accepted June 10, 1988.
