Detection of Cocaine and Its Major Metabolites in Rodent Bone Following Outdoor Decomposition after Chronic Cocaine Administration Using the ACQUITY UPLC System with 2D Technology

Significance of the Topic

Detection of drugs in skeletal remains addresses critical gaps in forensic toxicology when conventional matrices such as blood, urine, or soft tissue are unavailable or degraded. Bone offers a stable reservoir for chronic drug exposure markers, enabling retrospective reconstruction of substance use in decomposed or skeletonized specimens. This approach enhances the ability to link toxicological findings with pre-mortem drug administration in forensic and archaeological contexts.

Objectives and Study Overview

This work aimed to develop and validate a streamlined protocol for quantifying cocaine and its primary metabolites benzoylecgonine and ecgonine methyl ester in rodent bone after outdoor decomposition. Chronic cocaine dosing in rats, followed by controlled outdoor exposure for up to one year, provided skeletal specimens to assess the method’s sensitivity, recovery, and robustness. The study focused on optimizing extraction, clean-up, and two-dimensional UPLC-MS/MS analysis to achieve low-part-per-trillion limits of detection and high throughput.

Methodology and Procedure

• Sample preparation: 0.5 g of bone pulverized by high-speed impact homogenization with ceramic beads in methanol, followed by water re-extraction.
• Dilution and clean-up: Supernatants combined, filtered, then diluted into 100 mL of ultrapure water (final organic <5%) to prevent breakthrough.
• SPE cleanup: Oasis MCX mixed-mode cartridges (150 mg) conditioned and loaded under basic pH (pH 10), washed sequentially with acidified water and methanol/formic acid, then eluted with methanol containing 5% NH₄OH.
• Chromatography: Two-dimensional ACQUITY UPLC “trap & elute” configuration with at-column dilution. First dimension uses Oasis HLB trap at high pH, second dimension employs HSS T3 analytical column under a fast acetonitrile/formic acid gradient (10 min run).
• MS/MS detection: Xevo TQD triple quadrupole in positive ESI mode. Optimized MRM transitions for each analyte and deuterated internal standards ensure confirmation and quantitation.

Used Instrumentation

• Waters ACQUITY UPLC System with 2D Technology and at-column dilution
• Waters Xevo TQD triple quadrupole MS with MassLynx 4.1 software
• Oasis MCX SPE cartridges (60 mg and 150 mg sorbent mass)
• Ceramic bead homogenizer and high-speed centrifuge

Main Results and Discussion

• Limit of detection: 50 ppt (ng/L) for cocaine and 100 ppt for metabolites using 0.5 g bone.
• Extraction recoveries: ~85% for cocaine, ~72% for benzoylecgonine, ~54% for ecgonine methyl ester after matrix correction with deuterated internal standards.
• Linearity: Calibration in bone extract from 0.05–10 ppb for cocaine (R²=0.998) and 0.1–10 ppb for metabolites (R²≥0.997).
• Matrix effects: Ion suppression below 10% for all analytes, effectively compensated by internal standards.
• Application: Analysis of bones collected immediately post-mortem and after two years outdoors detected cocaine and benzoylecgonine at 0.01–0.20 ng/g, while ecgonine methyl ester frequently fell below quantification limits after extended exposure.
• Throughput: Sample preparation including homogenization and SPE in <60 minutes; UPLC-MS/MS cycle of 10 minutes per injection supports >1000 injections without performance loss.

Benefits and Practical Application

• Rapid workflow: Eliminates lengthy evaporation and reconstitution steps.
• High sensitivity: Achieves trace-level detection in challenging solid matrices.
• Robust clean-up: Mixed-mode SPE and 2D chromatography deliver clean extracts and stable baselines.
• Versatility: Adaptable to other drug classes, alternative hard tissues (hair, nails), and archaeological samples.
• Forensic utility: Provides reliable evidence in decomposed or skeletonized cases where traditional matrices are inaccessible.

Future Trends and Opportunities

• Extension to a broader panel of drugs and metabolites, including opioids and synthetic cannabinoids.
• Integration of high-resolution mass spectrometry for non-targeted screening in bone matrices.
• Automation of sample preparation steps and miniaturized SPE formats for enhanced throughput.
• Expansion to human forensic casework and archaeological studies to reconstruct historical drug exposure.

Conclusion

The developed micro-extraction and two-dimensional UPLC-MS/MS protocol enables rapid, sensitive, and robust quantification of cocaine and its metabolites in decomposed bone. By combining efficient homogenization, selective SPE clean-up, and advanced chromatography, the method addresses key challenges in forensic toxicology, offering a valuable tool for post-mortem investigations when conventional specimens are unavailable.

References

• Mallet CR, Botch-Jones S. J Anal Toxicol. 2016;40(8):1–11.
• Mallet CR. Multi-Dimensional Chromatography Compendium: Trap & Elute vs At-column Dilution. Waters Corp. 2015;720005339EN.
• Waters Application Brief. Analysis of Pharmaceuticals and Pesticides in Bottled, Tap and Surface Water Using ACQUITY UPLC with 2D Technology. 2014;720005167EN.
• Waters Corp. The Science of What’s Possible: ACQUITY UPLC, Oasis, XBridge, Xevo TQD. 2016;720005847EN.