Cocaine is one of the most widespread illicit drugs of abuse in the US. Cocaine N-oxide is a metabolite of cocaine formed by cytochrome P-450 mediated oxidation of cocaine.1,2 Cocaine N-oxide has been reported to be found in human plasma of cocaine users as well as meconium.1 Cocaine N-oxide isomers have been observed in human hair samples in varying amounts (personal communication, Dr. Mark Miller, FBI Quantico). Development of accurate methods to detect and quantitate cocaine N-oxide has applications in forensic / toxicology applications and in the screening for newborns when cocaine use in the parent is suspected.
Cocaine N-oxide is heat sensitive and readily degrades to cocaine and norcocaine on column when analyzed by GC/MS.1,2 Testing laboratories are developing LC/MS methods for this metabolite to monitor cocaine use in matrices such as hair. This poster presents the synthesis and stability of Certified Reference Materials (CRMs) of high purity cocaine N-oxide and its internal standard.
Cocaine N-oxide is a mixture of equatorial (anti) and axial (syn) isomers based on orientation of the N-oxide relative to substituents on the tropine ring.3
Cocaine N-oxide HCl was synthesized from cocaine HCl by oxidation with mCPBA in chloroform at 0 °C. Initial attempts to isolate pure cocaine N-oxide failed as the free base is unstable. Cocaine N-oxide was then converted to its hydrochloride salt in situ and purified to >98% (by HPLC), with an approximately 79/19 mixture of equatorial and axial isomers.
Cocaine N-oxide-D3 HCl was synthesized for use as an internal standard in a similar manner from cocaine-D3 HCl.
1H and 2D 1H-1H-COSY NMR were used to identify the two isomers.
The two isomers of cocaine N-oxide HCl were not stable enough to be individually isolated. 1H-NMR and 2D 1H-1H COSY spectra were obtained for the mixture of the isomers. The NMR spectra were measured in CDCl3 on a JEOL ECS-400 spectrometer. The1H-NMR shows two distinct sets of signals ~ 80:20, similar to the HPLC.
Cocaine N-oxide HCl and the internal standard were developed into ampouled solution-based Certified Reference Materials for use in identification and quantitation of cocaine use. The standard solution of cocaine N-oxide HCl was prepared at 1.0 mg/mL in acetonitrile. The internal standard was prepared at 100 μg/mL. Accelerated and real-time stability were performed for both native and labeled standards.
Accelerated and real-time stability of cocaine N-oxide HCl evaluated by HPLC is shown in Table 1
Cocaine N-oxide HCl CRM is stable under sub-freezer and freezer storage conditions for 1 year
Stability data for cocaine N-oxide-D3 HCl at 100 μg/mL in acetonitrile is shown in Table 2