Anal. Chem. 1986, 58,1349-1352
1349
High-Performance Liquid Chromatographic Chiral Stationary Phase Separation with Filament-On Thermospray Mass Spectrometric Identification of the Enantiomer Contaminant (S)-( +)-Methamphetamine Edgar D. Lee and Jack D. Henion* Drug Testing and Toxicology, New York State College of Veterinary Medicine, Cornell University, 925 Warren Drive, Zthaca, New York 14850
Charlotte A. Brunner, Irving W. Wainer, and Thomas D. Doyle* Center for Drugs and Biologics, Food and Drug Administration, 200
C Street, S.W., Washington, D.C. 20204
Joseph Gal Division of Clinical Pharmacology, University of Colorado School of Medicine, Denver, Colorado 80262
Enantlomerlc contamlnatlon at the 1% level can be detected and conflrmed by thermospray LC/MS techniques. A sample of methamphetamine contalnlng the enantlomers In a ratlo of 99:l was derlvatlzed wlth achiral 2-naphthyl chloroformate. The resultlng enantiomeric carbamates were well-separated ( R , = 2.07) on a Plrkle-type HPLC chlral stationary phase (CSP). Thermospray LC/MS detectlon In the "filament-on" mode demonstrated the structural ldentlty of the two enantlomerlc peaks by equlvalent Ion current proflles and relatlve abundances at m / z 320,228, and 176. Use of the CSP for separatlon of enantlomerlc solutes avolds problems In lowlevel detectlon Inherent In alternatlve methods such as those lnvolvlng formatlon of dlastereomers. “Fllamenton” capablllty for the thermospray Interface and MS detectlon provldes compatablllty wlth the normal phase HPLC systems required for many enantlomerlc separatlons on chlral stationary phases.
The positive identification of small amounts (ca. 1%)of one enantiomer in the presence of a preponderance of the other presents a unique analytical challenge. There are two analytical requirements for reliable detection and identification of enantiomers at this low level: (1) generation of discrete, well-resolved signals (e.g., chromatographic or spectroscopic) for both enantiomers which is difficult because of the intrinsic chemical and physical similarities of the isomers; and (2) unambiguous identification of the minor enantiomeric component, because the signal not only must be positively identified but also must be shown not to be an analytical artifact. These difficulties are well-illustrated by the problem of determining the stereochemical composition of methamphetamine (N-methyl-l-phenyl-2-aminopropane, desoxyephedrine, I). The dextrorotatory isomer, (S)-(+)-meth-
e
cH2-iH
--NH
-w3 111
C Ha amphetamine, is a controlled (Schedule 11) substance with a considerable history of drug abuse, whereas the levo isomer, (R)-(-)-methamphetamine, is the principal ingredient of an over-the-counter nasal decongestant. Differences in the hu0003-2700/86/0358-1349$01.50/0
man pharmacology and toxicology of the isomers are known ( I ) , but analytical differentiation of enantiomers has historically been a challenge. A number of analytical approaches have been developed that are satisfactory only when both isomers are present in appreciable amounts. Liu et al. employed a chiral NMR shift reagent to fully resolve the N-methyl resonances of (S)-(+)and (R)-(-)-methamphetamine (2),but sensitivity problems preclude detection of low-level amounts of one isomer. A more common approach has involved formation of diastereomeric derivatives employing chiral reagents followed by separation by chromatography on achiral columns. In this manner, the enantiomers of methamphetamine, and its primary amine analogue, amphetamine, were resolved by gas chromatography following reaction with L-proline (3) or with various analogues of this chiral reagent (4-7).High-performance liquid chromatography has been utilized for resolution of diastereomeric derivatives of methamphetamine formed by reaction with other analogues of proline. In a similar manner, chiral benoxaprofen was used as reagent prior to HPLC analysis of methamphetamine (9). These diastereomeric chromatographic methods afford from fair to excellent separation of (S)-(+)- and (R)-(-)-methamphetamine and all are quite sensitive with detection by UV or by mass spectrometry (10). However, all suffer from a serious conceptual and practical drawback in the detection of low levels of one enantiomer in the presence of the other. In diastereomeric analysis on an achiral column a peak attributable to the minor isomer of the anal@ may instead arise in whole or part from reaction of the major isomer with a small amount of enantiomeric contaminant of the (supposedly) pure chiral reactant. The RS’ diastereomer, for example, has chromatographic properties that are indistinguishable from the SR’diastereomer in an achiral system. This problem has been shown to be significant for reagents derived from proline and most of the modifications of this reactant were developed in an attempt to minimize racemization. Liu and Ku studied the consequences of chiral contamination of reactant in the analysis of amphetamine ( 1 1 ) and with others they extended this work to the determination of methamphetamine (12). Their method requires simultaneous analysis on both chiral and achiral GLC systems with algebraic correction for reagent contamination. However, since in the process of applying this approach they established that there was greater than 5 % enantiomeric contamination of the N-trifluoroacetyl-L-prolyl reactant, their results tend to em@ 1986 American Chemical Soclety
1350
ANALYTICALCHEMISTRY, VOL. 58, NO. 7, JUNE 1986
phasize the inherent uncertainty of this method for detection of low-level chiral components. In this paper we describe the separation and identification of (S)-(+)-and (E)-(-)-methamphetamine using an HPLC chiral stationary phase/thermospray mass spectrometric system. The methamphetamine sample is derivatized prior to chromatography with achiral2-naphthyl chloroformate (13, 14). The thermospray LC/MS system is operated under '%lament-on" conditions, which permits use of normal phase HPLC. This work is an extension of research in our laboratories on HPLC chiral stationary phase resolution of enantiomeric drugs (14,15) and on LC/MS thermospray interfaces (16) and MS identification of enantiomers (17). The utility of the method is shown by its application to analysis of a commercial inhaler of (R)-(-)-methamphetamine. The presence of 1% contamination by the (S)-(+)-* isomer was readily and conclusively demonstrated. EXPERIMENTAL SECTION Materials and Reagents. Authentic (S)-(+)-methamphetamine hydrochloride and racemic methamphetamine hydrochloride were obtained from Sigma (St. Louis, MO). HPLC grade hexane and acetonitrile were purchased from Burdick and Jackson (Muskegon, MI). The commercial inhaler was purchased locally and was labeled to contain 50 mg of 1-desoxyephedrine as the active ingredient, plus 150 mg of a mixture of menthol, camphor, methyl salicylate and bornyl acetate. The derivatizing reagent, 2-naphthyl chloroformate was prepared from 2-naphthol and phosgene by a modification of standard methods (15). All other reagenta and solvents were reagent grade. Preparation of Derivatives. Authentic (S)- or (SR)-methamphetamine hydrochloride (25 mg) was dissolved in 25 mL of water. The solution was made alkaline with 1M NaOH and then shaken for 5 min with a solution of approximately 100 mg of 2-naphthyl chloroformate in 25 mL of methylene chloride. The organic layer was then washed with water and dried with sodium sulfate prior to direct HPLC injection. (The solution may be optionally evaporated to dryness and reconstituted in methylene chloride without evidence of decomposition.) For the commercial inhaler, the entire inhaler contents was shaken for 5 min with 50 mL of 1M HaO,; 25 mL of this solution was washed with 25 mL of methylene chloride, the organic layer was discarded, and 25 mL of the acid layer was made alkaline with 1M NaOH. The solution was then derivatized as described above. HPLC Apparatus and Conditions. For the chromatography using only UV detection the system consisted of a Model SP 8700 pump, Model SP 8440 W-vis detector (set at 254 nm), and Model SP 4200 integrator (Spectra-Physics, Santa Clara, CA). For the LC/MS experisents the chromatographic system consisted of two Model 510 pumps, a Model 680 solvent programmer, a Model 440 UV detector with a fixed wavelength of 254 nm (Waters Associates, Milfrod, MA), and a Model 7520 injector with a 5-pL loop (Rheodyne, Cotati, CA). The column used in both systems was the identical 4.6 mm X 250 mm Bakerbond Chiral Phase DNBPG (covalent)column (J. T. Baker Co., Philipsburg, NJ). The column consists of (R)N-(3,5-dinitrobenzoyl)phenylglycinecovalently bonded to a 5-pm aminopropylsilica packing. The mobile phase was 98.5:1.5 hexane:acetonitrileoperated at ambient temperature. The flow rate was 1.5 mL/min. Mass Spectrometry. A Hewlett-Packard 5985B GC/MS equipped with a liquid-nitrogen-cooled cryopump and a Vestec thermospray LC/MS interface (Vestec,Inc., Houston, TX) was used. The exit of the HPLC detector was connected to the thermospray LC/MS probe via a Valco 0.5 pm in-line filter union (Valco Instrument Co., Houston, TX). (The cryopump is not considered essential in this system, but we find it facilitates routine operation of the system over prolonged periods.) The thermospray vaporizer temperature was maintained at 120 "C while the ion source region was held at 270 "C. RESULTS AND DISCUSSION Derivatization a n d Chromatography. The development
1
L Flgure 1. (A) HPLC chromatogram (with UV detection) of derivatized, authentic (R)-(-)- and (S)-(+)-methamphetamine, resolved on a Pirkle-type covalent CSP: (Peak 1) bis(2-naphthoyl) carbonate side product from reagent: (peaks 2 and 3) 2-naphthyl carbamate derivatives of (R)-(-)- and (S)-(+)-rnethamphetamlne, respectively. Chromatographic conditions and calculated parameters are given in the text. (B) Chromatogram of the derivatized extract of commercial inhaler labeled to contain (R)-(-)-methamphetamine, taken under identical conditions, and showing a minor component (peak 3) representing 1.3% of total methamphetamine.
and commercial availability of HPLC chiral stationary phases (CSPs) have made possible the routine, direct separation of enantiomers and avoids the problems inherent in the diastereomeric approach (13,14). Nevertheless, the enantiomers of methamphetamine and of other amine substances are not normally resolvable directly on the Pirkle-type phenylglycine CSP employed in this study. Formation of the 2-naphthyl carbamate derivative is necessary to improve chromatographic behavior and to confer enantioselectivity (15). Addition of the naphthyl chromophore also markedly increases sensitivity to UV detection. Since the reagent is achiral, no diastereomers are produced by the derivatization. The enantiomeric ratio of the products is in principle identical with that of the original sample, provided that the analytical conditions do not induce racemization of the sample itself. The absence of racemization was demonstrated in the present case by the chromatographic results for the reference sample (S)-(+)-methamphetamine. The amount of detectable (R)-(-)-isomer following conversion to the carbamate was less than 0.2%. This trace amount, if real, may have been present in the original sample. The results for the chromatography of the 2-naphthyl carbamates of (S)-(+)-and @)-(-)-methamphetamine are shown in Figure 1 (UV detection at 254 nm, without MS interface). Base line resolution was achieved even at an enantiomeric ratio of 99:l. The chromatographic parameters were as follows: capacity factors, 121' = 6.78, k i = 7.48; separation factor, 1.10;resolution, R, = 2.07; average number of plates, N = 7800 for the 25-cm column. Essentially symmetrical peaks were obtained by use of the hexane-acetonitrile mobile phase; this was at the expense of some loss of selectivity compared to the more commonly used mobile phase for this CSP of hexane-2-propanol. The selectivity and efficiency of the chromatographic system were more than sufficient to resolve the components of the commercial inhaler. As Figure 1 shows, the results indicated the possible presence of 1.3% of the (S)-(+)-enantiomer in the sample of (R)-(-)-methamphetamine.However, a t this low level there is an obvious need for confirmation of this result by positive identification of the minor peak. This identification was unambiguously provided by the mass spectrometric results. Mass Spectrometry. Thermospray LC/MS has received much recent attention (It?), and applications have proliferated as a result (16, 19, 20). One of the benefits afforded by
ANALYTICAL CHEMISTRY, VOL. 58, NO. 7, JUNE 1986
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1351
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Flgure 4. Filament-on thermospray LClMS TIC and extracted ion current profiles for m / z 320 (M l), 228,and 170 from (A) authentic sample of derivatlzed (R)-(-)- and (S)-(+)-methamphetamine and (B) derivatized extract of the commercial inhaler.
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thermospray is that total conventional HPLC effluents may be introduced directly into the mass spectrometer, including those containing high percentages of water. In fact, thermospray "filament-off" ionization requires high percentages of water in the eluent. This imposes a problem for chromatographers who wish to use eluents high in organic solvent or to perform analyses requiring normal phase HPLC conditions where water is absent from the eluent. Fortunately, modern thermospray LC/MS systems usually incorporate a conventional filament for so-called "filament-onn thermospray LC/MS. This ability to use an external source of ionization when thermospray or direct ionization (21)is not achieved extends the utility of thermospray LC/MS and is a necessary feature for anyone interested in a versatile LC/MS system. The requirements of the sample under study illustrate this versatility. Chiral LC/MS under the normal phase conditions required for most applications of Pirkle-type CSPs would have precluded thermospray LC/MS if "filament-on" capability were not available despite the evident added specificity of MS over a conventional UV detector. The results of the LC/MS detection are shown in Figures 2-4. The total ion current (TIC) (Figure 2B) for the "filament-on'! LC/MS analysis of the derivatized inhaler ex-
tract contains peaks 1 , 2 , and 3, corresponding to the three major components exhibited in the UV chromatogram (Figure 2A). The Y axis of this TIC has been expanded to aid visualization of the small chromatographic profile observed at a retention time of 12.6 min. Comparison of the UV chromatogram (Figure 2A) and the TIC shows a one-to-one correspondence between these two data sets. The "filament-on" thermospray LC/MS mass spectra Figure 3) provide some additional analytical specificity for the three peaks of interest beyond that shown in the UV and TIC. The abundant m/z 315 ion (Figure 3A) is consistent with an (M + 1) ion for the predicted byproduct bis(naphthoy1) carbonate, formed by dimerization of the reagent chloroformate. The two thermospray LC/MS spectra (Figure 3B,C) obtained from peaks 2 and 3 of the TIC show an abundant ion at mlz 320 with some additional less abundant ions present at m / z 176 and 228, and are identical in all respects (including retention time) with the peaks from the carbamate product of authentic (E+(+)- and (R)-(-)-methamphetamine. It should be noted that the mass spectrometer cannot distinguish between these two enantiomeric derivatives since it is an achiral device. The chromatographic chiral stationary phase provides the necessary chiral discrimination prior to mass spectrometry. However, precisely because the mass spectra of the two enantiomers are in principle identical in all respects, the isomer present in preponderance serves as the ideal reference standard for the minor component, providing by detailed comparison powerful confirmation of identity. For example, to provide additional confirmation that peak 3 from the commercial inhaler is in fact due to the suspect (9-(+)-methamphetamine isomer, the mass spectrometer data can be used to "extract" the ions characteristic of the analyte and present them (Figure 4)for comparison of both retention time and relative abundance. The extracted ion current profiles for ions characteristic of authentic (S)-(+)- and (R)-(-)-methamphetamine (Figure 4A) show the (M 1) ion at m/z 320 plus fragment ions of m / z 228 and 176 in addition to the TIC for comparison. These data reveal the coincidence
+
1352
Anal. Chem. 1986, 58, 1352-1355
of the ions for the 12.6-min component, which corroborates their unique origin. The corresponding extracted ion current profiles and TIC for the sample obtained for the inhaler (Figure 4B) show the same ions that are characteristic of (SI-(+)-and (R)-(-)methamphetamine ( m / z 320,228 and 176). Careful inspection of the extracted ion current profiles (Figure 4B) reveals a coincidence of retention time for the small ion current observed at 12.6 min, and a correspondence of relative abundance between the ions, as was observed for the authentic sample (Figure 4A). These results confirm the identity of the minor component as arising from low-level enantiomeric contamination of the sample.
CONCLUSION This work shows that detection and positive identification of enantiomeric contamination can be readily achieved down to a t least the 1% level. Facile and reliable analysis at this level is a result of advances in two areas: (1)the development of highly efficient methods for the resolution of enantiomers on HPLC chiral stationary phases, thus avoiding the introduction of errors from use of chiral reagents, and (2) the development of effective thermospray LC/MS interfacing with capability for “filament-on” detection, which allows use of normal phase chromatographic systems.
ACKNOWLEDGMENT E.D.L. and J.D.H. thank M. L. Vestal and Vectec Corp., for the generous loan of a thermospray LC/MS interface and power supply.
Registry No. (RS)-I, 4846-07-5;(S)-(t)-I, 537-46-2;@)-(-)-I, 33817-09-3; 2-naphthyl chloroformate, 7693-50-7.
LITERATURE CITED (1) @I, J. J . roxicoi. clin. roxicol. 1982, 19, 517.
(2) Liu, J. H.; Ramesh, H.; Tsay, J. T.; Ku, W. W.; Fitzgerald, M. P.; Ange10% S. A.; Llns, C. L. K. J . Forensic Scl. 1981, 26,656. (3) Wells, C. E. J . Assoc. Off. Anal. Chem. 1070, 53, 113. (4) Matin, S. B.; Rowland, M.; Castagnoli, J., Jr. J . Pharm. Sci. 1973, 62, 821. (5) Pohl, L. R.; Trager, W. F. J . Med. Chem. 1973, 16, 475. (6) Nichols, D. E.; Barfknecht, C. F.; Rusterholz, D. B.; Benington, R.; Morin, R. D. J . Med. Chem. 1973, 16, ‘480. (7) Gal, J. J . Pharm. Sci. 1077, 66, 169. (8) Barksdale, J. M.; Clark, C. R. J . Chromatogr. Sci. 1985, 23, 176. (9) Weber, H.; Spahn, H.; Mutschler, E. J . Chromatogr. 1984, 307, 145. (10) Liu, K.; Ku, W. K.; Fitzgerald, M. P.J . Assoc. Off. Anal. Chem. 1983, 66, 1443. (11) Liu, K.; Ku, W. K. Anal. Chem. 1981, 53,2180. (12) Liu, K.; Ku, W. K.; Tsay, J. T.; Fitzgerald, M. P.; Kim, S.J . Forensic Scl. 1982, 27, 39. (13) Pirkle, W. H.; Finn, J. M.; Schreiner, J. L.; Hemper, 8. C. J . Am. Chem. SOC. 1981, 103,3964. (14) Walner, I. W.; Doyle, T. D. LC Mag. 1984, 2 , 88. (15) Doyle, T. D.; Adams, W. M.; Fry, F. S.,Jr.; Wainer, I. W. J . Liquid Chromatogr ., in press. (16) Covey, T. R.; Crowther, J. B.; Dewey, E. A.; Henion, J. D. Anal. Chem. 1985, 56, 474. (17) Crowther, J. B.; Covey, T. R.; Dewey, E. A.; Henlon, J. D. Anal. Chem. 1984, 56, 2921. (18) Vestal, M. L. Science 1984, 226, 275. (19) Voyksner, R. D.; Bursey, J. T.; Hines, J. W. J . Chromatogr. 1985, 323,383. (20) Liberato, D. J.; Fenselau, C. C.; Vestal, M. L.; Yergey, A. L. Anal. Chem. 1983, 55, 1741. (21) Garteiz, D. A.; Vestal, M. L. LC Mag. 1085, 3 , 334.
RECEIVED for review January 9,1986. Accepted February 21, 1986.
Carboxymethylated Polyethylenimine-Polymethylenepolyphenylene Isocyanate Chelating Ion Exchange Resin Preconcentration for Inductively Coupled Plasma Spectrometry Zs. Horviith’ and Ramon M. Barnes* Department of Chemistry, GRC Towers, University of Massachusetts, Amherst, Massachusetts 01003-0035
A carboxymethylated polyethyienlmlne-polymethylenepolyphenylene Isocyanate chelating ion exchange resln was prepared, characterlred, and used for metals preconcentratlon for lnductlvely coupled plasma spectrometry. The uptake of copper, cadmlum, lead, and zinc by the resln was quantitative In the presence of high concentrations of ammonium, caklum, magneslum, potasslum, sodium, and acetate and citrate salts. These metals could be collected from artlficial seawater, Dead Sea water, and dissolved bone wlth a recovery of nearly 100%. The resln also chelates heavy metals and rare earths. Complexed metals can be eluted from the resin column wlth strong adds. The resln does not change volume with ionic form changes and can be regenerated for repeated use.
Chelating resins containing ligands with nitrogen and oxygen donor atoms on a polymer matrix are useful analytical Permanent address: L. Eotvos University, Institute of Inorganic and Analytical Chemistry, P.O. Box 123, H-1443 Budapest, Hungary.
reagents. These ligands are capable of forming a complex with a heavy metal atom incorporated into the polymeric material ( I ) . The best known of this type of resin is Chelex-100 (2), which has a polystyrene backbone and is widely used in trace analysis for preconcentration of heavy metals. However, Chelex-100 shrinks as its ionic form and pH change; for example, the resin swells 100% in changing from hydrogen to a monovalent salt form. Therefore, precautions such as wrapping columns with tape are required (2). The shrinkage is a drawback when the resin is used for collecting heavy metals from seawater (3). Dingman et al. (4) prepared polyamine-polyurea resins, and among them was a resin formed from polyethylenimine of molecular weight of 1800. A thorough study of the synthesis of these resins was reported by Hackett (5) and Hackett and Siggia (6). The resins were prepared by reacting polyethylenimine with molecular weight of 1800 (PEI-1800) with polymethylenepolyphenylene isocyanate (PAPI) to produce a cross-linked polyamine-polyurea polymer. The ratio of PEI/PAPI of 8 to 1,which was given in grams of the reactants, showed the best properties. This polymer was used to prepare
0003-2700/86/0358-1352$01.50/00 1986 Amerlcan Chemical Society
