The ability of bacterial cocaine esterase to hydrolyze cocaine metabolites and their simultaneous quantification using high-performance liquid chromatography-tandem mass spectrometry

Abstract

Cocaine toxicity is a widespread problem in the United States, responsible for more than 500,000 emergency department visits a year. There is currently no U.S. Food and Drug Administration-approved pharmacotherapy to directly treat cocaine toxicity. To this end, we have developed a mutant bacterial cocaine esterase (DM-CocE), which has been previously shown to rapidly hydrolyze cocaine into inert metabolites, preventing and reversing toxicity with limited immunogenic potential. Herein we describe the ability of DM-CocE to hydrolyze the active cocaine metabolites norcocaine and cocaethylene and its inability to hydrolyze benzoylecgonine. DM-CocE hydrolyzes norcocaine and cocaethylene with 58 and 45% of its catalytic efficiency for cocaine in vitro as measured by a spectrophotometric assay. We have developed a mass spectrometry method to simultaneously detect cocaine, benzoylecgonine, norcocaine, and ecgonine methyl ester to quantify the effect of DM-CocE on normal cocaine metabolism in vivo. DM-CocE administered to rats 10 min after a convulsant dose of cocaine alters the normal metabolism of cocaine, rapidly decreasing circulating levels of cocaine and norcocaine while increasing ecgonine methyl ester formation. Benzoylecgonine was not hydrolyzed in vivo, but circulating concentrations were reduced, suggesting that DM-CocE may bind and sequester this metabolite. These findings suggest that DM-CocE may reduce cocaine toxicity by eliminating active and toxic metabolites along with the parent cocaine molecule.

Fig. 1.

MS/MS spectra of cocaine and cocaine metabolites. Product ion MS/MS spectra were collected by direct infusion of a 5 to 10 μM solution of the authentic standards and deuterium-labeled internal standards using the optimized electrospray ionization source parameters listed in Table 1. The product ions selected for SRM transitions are indicated by an asterisk on the spectra. A, cocaine (COC) MS/MS spectrum; the SRM product ion is m/z 182.1. B, norcocaine (NOR) MS/MS spectrum; the SRM product ion is m/z 168.2. C, benzoylecgonine (BZE) MS/MS spectrum; the SRM product ion is m/z 168.3. D, ecgonine methyl ester (EME) MS/MS spectrum; the SRM product ion is m/z 82.0. Product ions selected for the deuterium-labeled internal standards are identical to the unlabeled standards except that the m/z values are shifted by 3 atomic mass units (amu).

Fig. 2.

Major cocaine metabolites. Cocaine is rapidly converted into several main metabolites. Human liver carboxylesterase-1 (hCE-1) demethylates 40 to 45% of cocaine to benzoylecgonine. hCE-1 also catalyzes the conversion of cocaine into the more potent metabolite cocaethylene when alcohol [ethanol (EtOH)]is present. Liver P450 CYP3A4 N-demethylates approximately 5 to 10% of cocaine to norcocaine. Serum butyrylcholinesterase (hBChE) (K_m = 14 μM, k_cat = 3.9 min⁻¹) (Xie et al., 1999) hydrolyzes 40 to 45% of cocaine into ecgonine methyl ester and benzoic acid. DM-CocE also catalyzes this reaction with a faster rate (K_m = 14 μM, k_cat = 1432 min⁻¹). DM-CocE hydrolyzes the hydrolysis of norcocaine to norecgonine methyl ester and benzoic acid (K_m = 29 μM, k_cat = 1307 min⁻¹) as well as that of cocaethylene to ecgonine ethyl ester and benzoic acid (K_m = 19 μM, k_cat = 1099 min⁻¹).

Fig. 3.

Hydrolysis of cocaine metabolites by DM-CocE. DM-CocE can hydrolyze the active cocaine metabolites cocaethylene and norcocaine but cannot hydrolyze benzoylecgonine, as determined by the spectrophotometric cocaine hydrolysis assay. Benzoylecgonine was not hydrolyzed; therefore, no data (n.d.) are reported in B to D. A, Michaelis-Menten saturation curves of DM-CocE hydrolysis of cocaine compared with those for the three major metabolites. B, the catalytic efficiency of DM-CocE for cocaethylene and norcocaine is 58 and 45%, respectively, that of cocaine (p < 0.001, Student's t test). C, the k_cat of DM-CocE for norcocaine was unchanged from the k_cat for cocaine. Cocaethylene had a significantly reduced k_cat as determined by Student's t test (p < 0.01). D, the K_m values of DM-CocE for both cocaethylene and norcocaine were significantly elevated above that for cocaine (p < 0.05 and p < 0.001, respectively).

Fig. 4.

LC-MS/MS analysis of cocaine and cocaine metabolites. Total ion chromatogram of a 4.00 μM calibration standard showing separation of ecgonine methyl ester (EME), benzoylecgonine (BZE), cocaine (COC), and norcocaine (NOR) on a Hypersil Gold ultra-performance liquid chromatography column (50 mm × 2.1 mm i.d., 1.9-μm particles) using 10 mM ammonium formate (pH 4.6) as solvent A and acetonitrile as solvent B at a flow rate of 0.45 ml/min. Then 4-μl aliquots were injected onto the column, and compounds were eluted over an 11-min gradient that began with an initial hold at 2% B for 1 min, increased to 18% B in the next minute, gradually ramped to 40% B over 8 min, and finally increased to 100% B in 1 min. Selected reaction monitoring was used for compound detection and quantification. The chromatogram was divided into three time segments during which only the relevant SRM transitions were scanned. This allowed for more accurate characterization of chromatographic peaks and, thus, more precise quantification.

Fig. 5.

LC-MS/MS calibration curves for cocaine and cocaine metabolites. Calibration curves were constructed for each compound by calculating the ratios of the LC-MS/MS peak areas of the target compounds to the corresponding deuterium-labeled internal standards and plotting the ratios as a function of analyte concentration normalized to the concentration of internal standard (IS). Data were fitted to a curve using least-squares linear regression analysis with 1/x weighting. The linear range for cocaine (COC), benzoylecgonine (BZE), and ecgonine methyl ester (EME) was 0.0313 to 4.00 μM. Because of the extremely low concentrations of norcocaine (NOR) observed in the in vivo studies, the calibration curve was adjusted correspondingly and ranged from 0.00313 to 0.400 μM. Regression coefficients (R²) were all ≥0.995 and accuracies typically ranged from 67 to 130%.

Fig. 6.

Hydrolysis of cocaine metabolites by DM-CocE in Sprague-Dawley rats. At time 0, 5.6 mg/kg cocaine is injected intravenously, followed 10 min later by either PBS or 0.32 mg/kg DM-CocE. Blood samples were taken throughout the time course and analyzed by mass spectrometry for cocaine metabolites simultaneously. A, cocaine is rapidly eliminated by DM-CocE. The concentration of cocaine rises again at t = 40 min because of the elimination of DM-CocE and redistribution of cocaine. B, benzoylecgonine concentrations are affected by DM-CocE, most likely because of binding and sequestration by DM-CocE. In addition, the absence of high concentrations of cocaine after 10 min and 45 s reduces the substrate for hCE-1 production of benzoylecgonine. C, ecgonine methyl ester concentrations are higher in DM-CocE-treated animals (equal to that of the original cocaine concentrations) demonstrating the production of EME by DM-CocE. D, norcocaine is rapidly eliminated by DM-CocE.