Yunming Ying, Brian Schulmeier, Kenan Yaser, Uma Sreenivasan, Kelley Huff, Beth Marek, Isil Dilek
Cerilliant Corporation, 811 Paloma Drive, Suite A, Round Rock, TX 78665
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