Rapid Analysis of Pharmaceuticals in Human Tissues Using the ACQUITY UPLC with 2D Technology

Importance of the Topic

Forensic toxicology relies on rapid, sensitive, and reliable methods to identify and quantify pharmaceuticals in postmortem human tissues. Accurate data on drug distribution in organs can determine cause and manner of death, support legal proceedings, and inform aviation accident investigations. Achieving trace-level detection in complex solid matrices remains challenging due to lengthy sample preparation and matrix interferences.

Objectives and Overview of the Study

This study aimed to develop and validate a fast, micro-scale extraction protocol combined with two-dimensional ultrahigh-performance liquid chromatography (2D UPLC) coupled to tandem mass spectrometry (MS/MS) for multi-residue analysis of 21 common drugs in human tissues. Key goals included:

• Reducing extraction time (< 45 min) without evaporation-to-dryness steps
• Achieving sub-ppt detection limits
• Minimizing matrix effects through optimized clean-up
• Demonstrating applicability to diverse tissues (brain, heart, lung, liver, kidney, spleen, stomach contents)

Methodology and Used Instrumentation

The workflow combined three main elements:

• Tissue homogenization: 1 g of tissue with 4 mL acetonitrile using ceramic ball milling at 6000 rpm (90 s), followed by centrifugation and filtration.
• Dual solid-phase extraction (SPE): Separate mixed-mode cartridges – strong cation exchange/reversed-phase (Oasis MCX) and weak cation exchange/reversed-phase (Oasis WCX) – to fractionate basic analytes from neutral/acidic interferences. Each cartridge was washed and eluted under optimized pH conditions, yielding two complementary fractions.
• Two-dimensional UPLC-MS/MS: ACQUITY UPLC with 2D Technology in trap-and-elute configuration, using an Oasis HLB trap column (40 mg) at neutral pH for loading and BEH C18 analytical column at 60 °C for separation. Xevo Q-TQ-S mass spectrometer in positive electrospray mode monitored two MRM transitions per analyte for quantification and confirmation.

Main Results and Discussion

2D LC optimization employed a 6×6 grid evaluating trap pH (3, 7, 10) and organic eluent composition. Method 6 (pH 7 loading onto 40 mg HLB, acetonitrile elution at pH 3) provided the best Gaussian peak shapes for all 21 drugs. SPE evaluation showed MCX alone yielded poor recovery for several analytes; combining MCX and WCX fractions ensured recoveries above 50% and matrix-matched recoveries between 75% and 110% for 18 compounds. Calibration was linear over 0.025–10 ng/mL (R2 > 0.995) with LODs of 0.001 ng/mL. Application to forensic case tissues detected dextromethorphan, flecainide, and citalopram at low ng/mL levels with minimal matrix suppression.

Practical Benefits and Applications

• Hands-on sample preparation completed in < 45 min without evaporation steps
• Trace-level quantification (ppt range) across diverse solid tissues
• High throughput: 10 min 2D UPLC run time, > 1,000 injections per column lifetime
• Robust performance in forensic toxicology, clinical research, and QA/QC of biological samples

Future Trends and Potential Applications

Advances may include automated, cartridge-based tissue homogenization, novel mixed-mode sorbents tailored for emerging psychoactive substances, miniaturization for single-cell analyses, and software-driven method development that further reduces development time. Integration with high-resolution mass spectrometry and ion mobility separations could enhance selectivity in highly complex matrices.

Conclusion

This work demonstrates a rapid, sensitive, and reliable micro-extraction coupled with 2D UPLC-MS/MS for multi-residue drug analysis in human tissues. The elimination of evaporation steps, the dual SPE approach, and optimized 2D chromatography provide excellent recoveries, trace-level detection, and robust performance suitable for forensic and clinical laboratories.

Used Instrumentation

• ACQUITY UPLC with 2D Technology (Waters Corporation)
• Xevo Q-TQ-S Tandem Quadrupole Mass Spectrometer
• MassLynx 4.1 Software
• Oasis MCX and WCX SPE Cartridges (Waters Corporation)
• Oasis HLB Trap Columns and BEH C18 Analytical Columns

References

1. The Scientific Working Group for Forensic Toxicology (SWGTOX). What is forensic toxicology? 2010.
2. Federal Aviation Administration. Forensic Toxicology Research Team activities. FAA Civil Aerospace Medical Institute.
3. Mallet CR, Botch-Jones SR. Automated 2D LC optimization for bio-analysis. J Anal Toxicol. 2016.
4. Mallet CR. Multi-Dimensional Chromatography Compendium: Trap and Elute vs AT-column Dilution. Waters App Note 720005339EN. 2015.
5. Mallet CR. Analysis of pharmaceuticals and pesticides in water using ACQUITY UPLC with 2D Technology. Waters App Note 720005339EN. 2014.