Anal. Chem. 2003, 75, 4335-4340
Detection and Time Course of Cocaine N-Oxide and Other Cocaine Metabolites in Human Plasma by Liquid Chromatography/Tandem Mass Spectrometry Shen-Nan Lin,*,† Sharon L. Walsh,‡ David E. Moody,† and Rodger L. Foltz†
University of Utah Center for Human Toxicology, 20S 2030E, Room 490, Salt Lake City, Utah 84112-9457, and Behavioral Pharmacology Research Unit, Department of Psychiatry and Behavioral Sciences, Johns Hopkins University School of Medicine, 5510 Nathan Shock Drive, Baltimore, Maryland 21224
Gas chromatography/mass spectrometry (GC/MS) is often used for detection and measurement of cocaine metabolites in biological specimens. However, cocaine N-oxide, a recently identified metabolite of cocaine, is thermally degraded when introduced into a GC/MS. The major degradation products are cocaine and norcocaine. When cocaine N-oxide was measured in rat plasma using liquid chromatography in combination with electrospray ionization-mass spectrometry (LC/ESI-MS), the cocaine N-oxide concentrations in the rat plasma were reported to be as high as 30% of the cocaine concentrations. However, in our study involving LC/ESI-MS/MS analysis of plasma collected from human subjects following administration of oral cocaine, we determined that the concentrations of cocaine N-oxide relative to the cocaine concentrations never exceeded 3%. This suggests that determination of cocaine concentration in human plasma by GC/MS analysis will not significantly distort the actual cocaine concentrations due to thermal conversion of cocaine N-oxide to cocaine. In the work reported here, we compared results obtained using GC/MS, LC/ESI-MS/MS, and liquid chromatography/atmospheric pressure chemical ionization-tandem mass spectrometry (LC/APCI-MS/MS) to determine thermal degradation of cocaine N-oxide. LC/ ESI-MS/MS was selected to determine cocaine, benzoylecgonine, and cocaine N-oxide, and LC/APCI-MS/MS was selected to determine ecgonine methyl ester and norcocaine in plasma collected from three human subjects participating in a clinical study. The resulting time course data provide additional information into kinetic interrelationships between cocaine N-oxidation and cocaine hydrolysis. Cocaine is capable of producing severe hepatocellular necrosis in laboratory animals and in humans. The mechanism of cocaine hepatotoxicity is not well understood but appears to be associated * Corresponding author. Phone: (801) 581-5117. Fax (801) 581-5034. E-mail:
[email protected]. † University of Utah Center for Human Toxicology. ‡ Johns Hopkins University School of Medicine. 10.1021/ac030037c CCC: $25.00 Published on Web 07/19/2003
© 2003 American Chemical Society
with actions of one or more N-oxidative metabolites of cocaine.1-3 Norcocaine, the most prominent N-oxidative metabolite, is reported to be formed by two alternative routes: (1) direct N-demethylation by cytochrome P450 and (2) formation of cocaine N-oxide by FAD-containing monooxygenase, followed by cyctochrome P450-mediated conversion to norcocaine and formaldehyde.4 Cocaine N-oxide has been detected in rat plasma5 and in human meconium.6 Liquid chromatography/electrospray ionization-mass spectrometry (LC/ESI-MS) was required for those studies because cocaine N-oxide is converted back to cocaine when samples are analyzed by gas chromatography/mass spectrometry (GC/MS). Cocaine N-oxide concentrations in rat plasma were reported as high as 30% of the cocaine concentration measured by LC/MS.5 Those observations have raised concerns regarding the accuracy of cocaine measurements in human plasma that are based on GC/MS analysis, because such a high percentage of cocaine N-oxide would cause cocaine concentrations to be overestimated. Cocaine N-oxide has not been previously reported in human plasma. In this study, we compared thermal stability of cocaine N-oxide under GC/MS, liquid chromatography/electrospray ionization tandem mass spectrometry (LC/ESI-MS/MS), and liquid chromatography/atmospheric pressure chemical ionization-tandem mass spectrometry (LC/APCI-MS/MS) conditions. Less than 0.5% of cocaine N-oxide decomposition occurred during LC/ESI-MS/ MS analysis. Therefore, LC/ESI-MS/MS was selected for simultaneous determinations of cocaine N-oxide, cocaine, and benzoylecgonine in human plasma samples collected at multiple time points after oral administration of cocaine. Concentrations of norcocaine and ecgonine methyl ester in the same plasma samples (1) Ndikum-Moffor, F. M.; Schoeb, T. R.; Roberts, S. M. J. Pharmacol. Exp. Ther. 1998, 284, 413-419. (2) Boess, F.; Ndikum-Moffor, F. M.; Boelsterli, U. A.; Roberts, S. M. Biochem. Pharmacol. 2000, 60, 615-623. (3) Lloyd, R. V.; Shuster, L.; Mason, R. P. Mol. Pharmacol. 1993, 43, 645648. (4) Kloss, M. W.; Rosen, G. M.; Rauckman, E. J. Mol. Pharmacol. 1983, 23, 482-485. (5) Wang, P. P.; Bartlett, M. G. J. Anal. Toxicol. 1999, 23, 62-66. (6) Xia, Y.; Wang, P. P.; Bartlett, M. G.; Solomon, H. M.; Busch, K. L. Anal. Chem. 2000, 72, 764-771.
Analytical Chemistry, Vol. 75, No. 16, August 15, 2003 4335
were determined in separate runs using a modification of our validated LC/APCI-MS/MS assay.7 The data allow kinetic analysis of the metabolic interrelationships of cocaine N-oxide with cocaine and other oxidative and hydrolytic metabolites. EXPERIMENTAL SECTION Materials. Cocaine, cocaine-d3, benzoylecgonine, benzoylecgonine-d3, norcocaine, norcocaine-d3, ecgonine methyl ester, and ecgonine methyl ester-d3 were purchased from Radian International (now Cerilliant, Austin, TX). Cocaine N-oxide was provided by the National Institute on Drug Abuse (Bethesda, MD). Clinical Specimens. Blood was collected from three subjects taking part in a larger study concerning the time course, nature, and magnitude of withdrawal from repeated cocaine use. All subjects were healthy individuals with prior history of cocaine use. At the time of the study, they were residing in the Behavioral Psychology Research Unit at the Johns Hopkins University for a period of at least 40 days. Samples were collected after the first of a series of oral administrations of cocaine. Within a session, the dosing and blood collection protocols were similar to those previously described by Jufer et al.8 from a separate dose-ranging study. Cocaine (175 mg, po) was administered at 0, 1, 2, 3, and 4 h (total 875 mg, po, over 5 h). Blood was collected by venipuncture into gray-topped Vacutainer tubes just before the initial administration and then at 0.5, 1, 2, 3, 4, 5, 6, 7, 8, 12, and 24 h. Each blood sample was centrifuged soon after collection, and the plasma fraction was transferred to a separate container and stored at -20 °C. Samples were shipped on dry ice to the University of Utah and retained at -20 °C until analysis. Extraction. Deuterium-labeled isomers (25 ng each of cocained3, benzoylecgonine-d3, norcocaine-d3, and ecgonine methyl esterd3) were added as the internal standards to 1-mL aliquots of plasma specimens and calibrators. Calibrator samples for LC/ESI-MS/ MS analysis were freshly prepared just prior to extraction by spiking a series of 1-mL blank plasma samples with cocaine, benzoylecgonine, and cocaine N-oxide to give concentrations of 2.5, 5, 10, 30, 90, 350, and 750 ng/mL. Calibrator samples for LC/ APCI-MS/MS analyses were prepared by replacing the cocaine N-oxide with norcocaine and ecgonine methyl ester. Plasma samples were then extracted using our previously described solidphase extraction method for cocaine and benzoylecgonine.7 GC/MS. To investigate thermal degradation of cocaine N-oxide under GC/MS conditions, cocaine N-oxide was dissolved in ethyl acetate and analyzed using a HP5973 MSD with a HP6890 series GC. The capillary column was a DB5, 30 m × 0.25 mm i.d. with 0.25-µm film thickness (J&W, Folsom, CA). Helium was the carrier gas. The oven temperature was programmed starting from 140 °C for 1 min and then increased to 250 °C at 18 °C/min and held at 250 °C for 2 min. The injector temperature was 250 °C (unless stated otherwise). Sample molecules were subjected to positive ion chemical ionization (PCI) using methane as the reagent gas, and ions were detected by selective ion monitoring (SIM). LC/ESI-MS/MS and LC/APCI-MS/MS. Injections were made using a CTC-A200S autosampler (Finnigan MAT, San Jose, CA) into a Waters 626 liquid chromatograph (Milford, MA) with (7) Lin, S.-N.; Moody, D. E.; Bigelow, G. E.; Foltz, R. L J. Anal. Toxicol. 2001, 25, 497-503. (8) Jufer, R. A.; Walsh, S. L.; Cone, E. J. J. Anal. Toxicol. 1998, 22, 435-444.
4336
Analytical Chemistry, Vol. 75, No. 16, August 15, 2003
an Inertsil 5-µm particle size ODS-AQ (100 mm × 2 mm) LC column (YMC Waters, Milford, MA). The mobile phase consisted of 55% water containing 0.1% formic and 45% methanol with a flow rate of 0.15 mL/min. Mass spectrometry was performed using a Finnigan model TSQ-7000 triple quadrupole mass spectrometer with an ESI or an APCI interface to the liquid chromatograph. Conditions for ESI operations included a heated capillary temperature of 225 °C (unless stated otherwise), an electrospray voltage of 4.5 kV with the sheath gas pressure set at 60 psi, and auxiliary gas of nitrogen gas at the arbitrary unit of 12 on the flow meter associated with the TSQ-7000 mass spectrometer. Conditions for APCI operations included a heated capillary temperature of 150 °C, a thermal evaporator temperature of 375 °C (unless stated otherwise), and a corona source current of 4.5 µA with the nitrogen sheath gas pressure set at 30 psi. Cocaine, cocaine-d3, benzoylecgonine, benzoylecognine-d3, cocaine N-oxide, norcocaine, norcocaine-d3, ecgonine methyl ester, and ecgonine methyl ester-d3 were detected by selective reaction monitoring (SRM) with a collision energy of 20 eV and a collision gas (Ar) pressure of 2.6-3.0 mTorr. Calibration curves were established from the peak area ratios (cocaine/cocaine-d3, benzoylecgonine/benzoylecgonine-d3, cocaine N-oxide/cocaine-d3, norcocaine/norcocaine-d3, or ecgonine methyl ester/ecgonine methyl ester-d3) of calibrators from 2.5 to 750 ng/ mL, using linear regression with a 1/Y2 weighting. RESULTS AND DISCUSSION GC/MS of Cocaine N-Oxide. Wang and Bartlett have demonstrated that cocaine N-oxide in 1 µL of a 1 µg/mL concentration was 100% thermally degraded during GC/MS analysis, and cocaine was the only reported degradation product.5 In our study, we also observed that cocaine N-oxide in 1 µL of a 5 µg/mL concentration was undetectable by GC/MS analysis. Furthermore, in addition to cocaine as degradation product, we found that norcocaine was formed in substantial quantities at lower injector temperatures (
