Anal. Chem. 1984, 56, 2921-2926
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Liquid Chromatographic/Mass Spectrometric Determination of Optically Active Drugs J. B. Crowther, T. R. Covey, E. A. Dewey, and J. D. Henion* Equine Drug Testing and Toxicology Program, New York State College of Veterinary Medicine, Cornel1 University, 925 Warren Drive, Ithaca, New York 14850
Commercially available bonded chirai statlonary-phase HPLC columns (CSP-HPLC) were used to resolve optical isomers of ibuprofen benzylamide, N-1-naphthoyiamphetamine, fenoprofen 1-naphthalenylmethylamide, and benoxaprofen 1naphthalenylmethylamide. Comparisons of UV chromatograms of synthetic standards were made with ion current profiles obtained from split effluent direct liquid introduction (DLI) LC/MS of these compounds. DLI LC/MS results on equine urine extracts obtained from a 2-h postadministration ibuprofen sample were obtained. The (S )-(+)-ibuprofen enantiomer was the predominant species in both a TLC scrape of a 2-h equine urine extract and the crude 2-h equlne urine extract. An unknown component coeiuting with the ( R ) (-)-ibuprofen enantiomer was detected by DLI-LC/MS which was not detected by UV detection.
Stereoselectivity is an important phenomenon in biological systems. In most cases optical enantiomers exhibit different biological activities due to their specific interaction with the corresponding receptor, different transport mechanisms, or metabolic pathways ( I ) . Drugs which are characterized by these biological differences include ephedrine, epinephrine, phenylephrine, and others (2). For example, it is the d-enantiomer of penicillamine that is effective in cases of cystinuria and mercury poisoning, but the 1-enantiomer of Nphthaloylglutamic acid, a metabolite of thalidomide, is teratogenic (4). The stereoisomers of popular nonsteroidal antiinflammatory agents such as ibuprofen and naproxen show significant differences in potency and metabolism (5). Thus, it is important to be able to distinguish and identify optical isomers. The classical method of measuring the specific rotation of one enantiomer of a racemic mixture is not appropriate for modern drug metabolism studies. Typically the level of isolated drug or metabolite is far too low (5) and exists in the presence of high levels of interfering components. Thus, sensitive detection methods and highly efficient chromatographic capability are needed to resolve such analytical problems. Recent gas chromatographic analyses of biological samples containing optically active compounds have been performed in two ways: by derivatization with a pure enantiomer of an optically active reagent and separation of the resulting diastereomers on a nonchiral stationary phase (6, 7 ) or by direct separation of the enantiomers on a chiral stationary phase (8). Similarly, recent advances in the resolution of stereoisomers with chiral stationary phases and high-performance liquid chromatography (CSP-HPLC) have provided the capability for analysis of enantiomeric compounds using conventional chromatographic techniques (9,10). In particular, application of CSP-HPLC to pharmaceutical analysis of amide derivatives of 1-phenyl-2-aminopropanehas shown impressive successes (11).
The simplest mode of distinguishing enantiomers would be the direct determination of the underivatized compounds by the chromatographic process. Hermansson (12) has recently demonstrated an application of this capability, but much of the related work reported thus far has utilized selected derivatives which have been found to improve solute/support interactions (10, 11). Wainer and Doyle (13)have stressed the value of preparing suitable enantiomeric derivatives from achiral reagents. The latter forms simple amide derivatives, for example, which preclude enantiomeric contamination, differing reaction rates and/or equilibrium constants, and unsatisfactory chromatographic performance. The commerical availability of chiral columns and ease of amide-forming derivatization procedures for certain compound classes make CSP-HPLC a convenient method of resolving and distinguishing some biologically important enantiomers. Unfortunately, simply comparing HPLC retention times of resolved standards and unknown enantiomeric derivatives does not provide unequivocal identification of optical isomers. A detection system with greater specificity than UV or fluorescence detection is required to distinguish enantiomeric derivatives from unknown interfering components. This is particularly important for unknown biological samples such as plasma and urine extracts. In certain cases we have used combined high-performance liquid chromatography/mass spectrometry (LC/MS) (14-16) to differentiate between the enantiomers of administered drugs in race horses. The direct liquid introduction (DLI) technique of LC/MS (17) is readily amenable to CSP-HPLC analyses as reported by Pirkle (10) and Wainer (11). This paper reports the results of LC/MS using a chiral stationary phase (CSPLC/MS) on a series of enantiomeric carboxylic acids and, in particular, the detection and identification of (S)-(+)-ibuprofen in equine urine after administration of (S,R)-ibuprofen.
EXPERIMENTAL SECTION Materials. HPLC grade hexane and isopropyl alcohol were purchased from Burdick and Jackson (Muskegon, MI). Thionyl chloride and benzylamine were purchased from Aldrich Chemical Co. (Milwaukee, WI) and used without further purification. Ibuprofen (Motrin) for administration and use as an analytical standard was obtained from The Upjohn Co. (Kalamazoo, MI). The authentic amide derivatives of d,l-amphetamine, (R,S)-fenoprofen, (R,S)-benoxaprofen,and (R,S)-ibuprofenwere generously provided in pure, cystalline form from I. W. Wainer and T. D. Doyle (FDA, Washington, DC). HPLC Apparatus and Conditions. The HPLC equipment consisted of two Waters M-6000A pumps and a M-660 solvent programmer (Waters Assoc.; Milford, MA). The injector was a Rheodyne Model 7520 microloop injector with a 5-gL loop (Rheodyne,Cotati, CA). The CSP columns used were covalently (DNBPG) Pirkle bound (R)-N-(3,5-dinitrobenzoyl)phenylglycine Type 1A chiral stationary phase purchased from Regis Chemical Co. (Morton Grove, IL) and J. T. Baker Chemical Co. (Phillipsburg, NJ). The CSP columns were both stainless steel 4.6 mm X 25 cm packed with 5 - ~ m spherical support which were connected to a Waters Model 440 UV detector equipped with a standard
0003-2700/84/0356-2921$01.50/0 0 1984 American Chemical Society
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ANALYTICAL CHEMISTRY, VOL. 56, NO. 14, DECEMBER 1984
flow cell and operated at 254 nm. The eluent was 95/5/1 hexane/isopropyl alcohol/acetonitrile or 9713 and 92/8 hexane/ isopropyl alcohol (v/v) (see text) maintained at an isocratic flow rate of 2.0 mL/min. All experiments were conducted with the CSP column at ambient temperature. The UV chromatographic trace was recorded on a Fisher Recordall series 5000 (Fisher Scientific Co, Pittsburgh, PA) recorder operated at 10 mV and a chart speed of 20 cm/h. Subject and Drug Administration. Ibuprofen, 2 g, was administered orally to adult mares. Urine samples were collected prior to drug administration and at 2-h intervals postadministration. Samples were stored frozen until analyzed. Extraction and Isolation Procedure. Postadministration equine urine (9 mL) was adjusted to pH 3.5 by dropwise addition of 6 N HC1. The acidified urine was extracted by mixing on a rotorack for 10 min with a 5-mL portion of hexane/dichloromethane/diethyl ether (l/l/l).The organic layer was transferred and evaporated to dryness under a gentle stream of nitrogen in a 65 "C water bath. This residue is referred to later as the crude urine extract. Ibuprofen was isolated from this residue by preparative thinlayer chromatography (TLC). An ethyl acetate solution of the residue was applied as a band to silica gel 60 F,, TLC plates 0.25 mm thick (EM Science, Darmstadt, Germany). The plates were developed to a height of 5 cm in chloroform/methyl alcohol (9/1), Ibuprofen was located by observation of slight quenching under short wavelength UV light at the same Rfas standard ibuprofen (0.61). Ibuprofen may be visualized by spraying a portion of the TLC plate with Mandelins reagent (0.5 g of ammonium vanadate dissolved in 100 mL of concentration H,SO,) followed by heating the TLC plate. After the desired component was located, the appropriate area was scraped from the plate. The silica gel was added to a standard 15-cm Pasteur pipet plugged with a 2-mm length of porous polypropylene which had been prewashed with methyl alcohol. Ibuprofen was eluted from the silica gel with methyl alcohol. The eluent was concentrated under a gentle stream of nitrogen in a 65 "C water bath and is later referred to as the TLC scrape. A control experiment using authentic racemic ibuprofen was carried out exactly as described above to verify that achiral TLC does not resolve the ibuprofen enantiomers. A preparative TLC scrape of racemic ibuprofen was analyzed by CSP-HPLC. The CSP-HPLC UV chromatogram obtained from the TLC scrape was identical with that obtained from the standard racemic mixture of ibuprofen. Thus, the preparative TLC sample clean-up step does not resolve the enantiomers of ibuprofen. Mass Spectrometer. A Hewlett-Packard 5985B GC/MS equipped with a liquid nitrogen cryopump and the HewlettPackard split effluent DLI LC/MS interface (Option 01, Hewlett-Packard Co., Palo Alto, CA) was used in this work. The exit of the Waters 440 UV detector was connected to the DLI LC/MS interface via a Valco 0.5-pm in-line filter union (Valco Instrument Co., Houston, TX). At the operating HPLC eluent flow rate of 2.0 mL/min, the fine metering valve on the LC/MS interface was adjusted to provide about 40 pL/min effluent introduction into the mass spectrometer. About 2% of the total LC effluent is introduced into the ion source and results in reduced overall LC/MS detection limits. The mass spectrometer was operated in the positive ion chemical ionization mode (PCI) using the LC eluent 97/3 or 92/8 hexane/isopropyl alcohol as the CI reagent gas. The electron ionization energy was 230 eV at 300-pA emission current with the standard CI source block maintained at 250 "C. The standard electron focusing magnet ring was removed to allow use of the extended desolvation chamber described previously (18). Full scan acquisition included a scan from m / z 200 to m / z 500.
RESULTS AND DISCUSSION The structures of the compounds studied in this work are shown in Figure 1. These types of compounds are of interest in the drug testing of race horses and have been studied using CSP-HPLC by others ( I I ) . Preliminary experiments consisted of establishing satisfactory separation of synthetic standards of a variety of authentic amide derivatives by CSP-HPLC with UV detection in the off-line mode. We found adequate sep-
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aration of a variety of naphthamide and benzylamide derivatives of (R,S)-methylarylacetic acid antiinflammatory agents and d,l-amphetamine. Figure 2 shows the corresponding UV chromatograms of standard (R,S)-ibuprofen benzylamide using a Regis-packed, PirMe Type 1A (4.6 mm x 25 cm) CSP-HPLC column (Figure 2B) and J. T. Baker Bakerbond DNBPG (4.6 mm X 25 cm) (Figure 2A). The eluent composition in each case was 2.0 mL/min 951511 hexane/isopropyl alcohol/acetonitrile delivered at 2.0 mL/min. All other experimental variables were maintained constant. Although adequate separation is accomplished in each case, Figure 2A shows that the J. T. Baker column provides adequate separation in about half the time. This appears to be a general phenomenon which has been experienced by others (19). Thus, to reduce LC/MS run times and minimize exposure of the ion source to excessive LC effluent, the LC/MS analyses of urinary extracts were performed with the J. T. Baker CSP column. It should be noted, however, that when the eluent was adjusted to produce the same k' values on each column, the J. T. Baker and Regis-packed Pirkle columns gave comparable results. When satisfactory CSP-HPLC separations had been obtained from a variety of authentic samples of R,S-enantiomeric amides, the HPLC system was connected to the DLI LC/MS
ANALYTICAL CHEMISTRY, VOL. 56, NO. 14, DECEMBER 1984 A
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ANALYTICAL CHEMISTRY, VOL. 56, NO. 14, DECEMBER 1984
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Flgure 6. (A) UV chromatogram at 254 nm of 1 pg of an authentic mixture of the 1-naphthalenylmethylamide of (R ,S)-benoxaprofen. Chromatographic conditions were the same as those described in Figure 3A. The two major chromatographic peaks centered at about 22 min are (S)-(+)- and (R)-(-)-benoxaprofen 1-naphthalenylmethylamide, respectively. (B) On-line CSP-LC/MS extracted ion current profile for the (M + 1) ion of the sample described in (A).
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adequate chromatographic peak shape is accomplished within 30 min. In Figure 4B the total ion current profile and EICP for the protonated molecular ion a t m / z 382 and a major fragment ion at m/z 242 is shown for an on-column injection of 5 pg of authentic (R,S)-fenoprofen 1-naphthalenylmethylamide. A comparison of the ion current profiles and the UV trace in Figure 4 demonstrates similar chromatographic results between the UV and mass spectrometer detectors. The time scales in Figure 4 do not correspond because LC/MS data acquisition commenced approximately 20 min after injection of the sample. A comparison of the PCI LC/MS mass spectra shown in Figure 5 reveals the similarity of the mass spectra obtained from the (R,S)-fenoprofen amides. The corresponding total ion current profile in Figure 4B shows the elution of (R,S)fenoprofen amide a t 5.12 and 7.6 min, respectively. In addition, several structurally significant fragment ions in each spectrum of Figure 5 provide the information necessary for the identification of these compounds in an unknown sample. In a similar fashion we show the UV and LC/MS ion current traces for the chiral separation of a R,S mixture of standard benoxaprofen 1-naphthalenylmethylamide (Figure 6) and ibuprofen benzylamide (Figure 7). In each case the corresponding ion current profiles are comparable to the UV chromatogram. However, the unfavorable HPLC split of the effluent in the DLI LC/MS interface provides a weak ion current trace in Figure 6B for 1 pg of (R,S)-benoxaprofen 1-naphthalenylmethylamideinjected on-column. However, the total ion current profile for 10 bg of (R,S)-ibuprofen benzylamide injected on-column shown in Figure 7B provides
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Flgure 7. (A) UV chromatogram at 254 nm of 10 pg of an authentic mixture of the benzylamide of (R,S)-ibuprofen. The CSP column was a J. T. Baker packed Pirkle covalent bound (R)-N-(S,B-dinitrobenzoy1)phenylglycine 4.6 mm X 25 column operated at 2.0 mL/min hexane/isopropyl alcohol (9218). The two major chromatographic peaks centered at about 7 min are (S)-(+)- and @)-(-)-ibuprofen benzylamide, respectively. (B) On-line CSP-LCIMS TIC of the sample described in (A). a strong, stable ion current trace. In each case the corresponding UV traces displayed a high signal-to-noise ratio because all the sample passed through the UV detector. The 98% split of the HPLC effluent away from the ion source is necessary because of the limitations of the vacuum system. When microbore CSP-HPLC columns (1-mm i.d.) become available, the reduced eluent flow rates utilized with these columns should allow total micro LC effluent introduction into the mass spectrometer. This should provide significantly improved micro LC/MS detection limits for resolved R,Senantiomeric pairs. Since the ultimate test of any analytical technique is its application to real-world samples, we have used this CSPLC/MS system for the determination of ibuprofen in equine urine. The preparative TLC scrape (see Experimental Section) was derivatized to form the benzylamide according to the procedure of Wainer and Doyle (11) and analyzed by CSP-LC/MS. The resulting ion current profile is shown in Figure 8B and compared with the corresponding data obtained from an authentic mixture of (R,S)-ibuprofen benzylamide shown in Figure 8A. LC/MS data acquisition commenced with sample injection so that UV and mass spectrometer
ANALYTICAL CHEMISTRY, VOL. 56, NO. 14, DECEMBER 1984 **
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