Crocodile Tears: The Metabolic Fate and Detection of Desomorphine

The opioid desomorphine is a morphine alternative often abused as “crocodile” due to its inexpensive nature relative to heroin. Crocodile synthesis requires codeine plus readily available chemicals. The latter also produce characteristic side effects, including damage to the muscle, bone and vasculature at the injection site, sometimes leading to tissue necrosis and gangrene.

Currently, no information is available regarding the metabolic fate of desomorphine and its detectability using standard urine screening. Since metabolites make excellent targets for urinalysis, a complete investigation of desomorphine metabolism could assist researchers in establishing a marker for crocodile use and/or opioid poisoning.

To this end, Richter et al. (2016) turned to in vivo investigation via rat urine and in vitro investigation using pooled human liver microsomes (pHLM), pooled human liver cytosol (pHLC) and human liver cell lines (HepG2 and HepaRG).¹ For this, they used two mass spectrometric instruments/approaches:

1. Orbitrap-based liquid chromatography–high-resolution tandem mass spectrometry (LC-HR-MS/MS), using an Accela LC system coupled with a Q Exactive mass spectrometer (Thermo Scientific) and controlled by Xcalibur software (Thermo Scientific)
   

2. Hydrophilic interaction LC-HR-MS/MS, using an UltiMate 3000 rapid separation system and a Q Exactive Plus Hybrid Quadrupole-Orbitrap mass spectrometer with a heated electrospray ionization source (all Thermo Scientific)
   

The team characterized phase I and phase II metabolites of desomorphine, producing a detailed schematic of its metabolic fate, including HR-MS/MS spectra, proposed structures and retention times, as well as precursor ions and fragment ions with measured accurate mass and mass deviation calculations. For phase I metabolites, the research team identified a single catalyzing agent: CYP3A4. For phase II metabolites, they detected multiple enzymes catalyzing glucuronidation: UGT1A1, UGT1A8, UGT1A9, UGT1A10, UGT2B4, UGT2B7, UGT2B15 and UGT2B17. The researchers note that UGT2B7 also serves as the main catalyzing agent for glucuronidation of morphine.

They detected the following desomorphine metabolites by model:

Table 1. Phase I metabolites

Metabolite	Present in
Nor	All models
Hydroxy isomer 1 (dihydromorphine)	Rat urine only
Hydroxy isomer 2	All models
Hydroxy isomer 3	Rat urine, HepG2, HepaRG
Hydroxy isomer 4	Rat urine, pHLM/pHLC, HepaRG
Hydroxy isomer 5	pHLM/pHLC
N-oxide	All models

Table 2. Phase II metabolites

Metabolite	Present in
Glucuronide	All models
Sulfate	Rat urine, HepG2, HepaRG
Nor glucuronide	Rat urine, HepaRG
N-oxide glucuronide	Rat urine, HepaRG

Richter et al. report that for the in vivo model (rat urine), the most abundant peak was desomorphine glucuronide, followed by desomorphine. When compared with the in vitro models, the peak areas for metabolites observed here were lower in relation to desomorphine. The researchers indicate that the metabolites tended to accumulate in the wells or reaction tubes, limiting their formation.

All in vitro models identified the most abundant metabolite, desomorphine glucuronide. The HepaRG model produced higher peak area ratios for both phase I and phase II metabolites as well as a greater number of different metabolites. The research team posits that this model could serve as an in vitro tool for predicting hepatic metabolism of drugs of abuse but call for further studies to confirm this, including in vivo data like clinical and forensic casework.

Finally, Richter et al. report the following MS detections in rat urine following administration of a 1 mg/kg body weight dose of desomorphine:

• Desomorphine and its nor metabolite by gas chromatography–MS

• Desomorphine and its glucuronide by LC–multiple-stage MS

• Desomorphine, its nor metabolite and its glucuronide by LC-HR-MS/MS

After scaling, this dose corresponds with a 0.2 mg/kg human dose. Since the common therapeutic dose is up to 60 mg (1 mg/kg body weight), the team indicates that abuse should be detectable by standard urine screening, assuming comparable human kinetics.

Overall, the team offer this data as a detailed picture of the metabolic fate of desomorphine. They also propose in vitro liver cell culture (HepaRG cells) as potentially suitable as a tool for predicting hepatic metabolism of drugs of abuse and demonstrate the detectability of desomorphine and its glucuronide by MS-based urinalysis.

Reference
1. Richter, L.H., et al. (2016) “Metabolic fate of desomorphine elucidated using rat urine, pooled human liver preparations, and human hepatocyte cultures as well as its detectability using standard urine screening approaches,” Analytical and Bioanalytical Chemistry, 408(23) (pp. 6283–6294), doi: 10.1007/s00216-016-9740-4.

Post Author: Melissa J. Mayer. Melissa is a freelance writer who specializes in science journalism. She possesses passion for and experience in the fields of proteomics, cellular/molecular biology, microbiology, biochemistry, and immunology. Melissa is also bilingual (Spanish) and holds a teaching certificate with a biology endorsement.