Quantification of 78 drugs of abuse in human blood by liquid chromatography – HRAM Orbitrap mass spectrometry for clinical research

Importance of the Topic

A growing variety of designer and synthetic drugs of abuse pose significant challenges for clinical and forensic laboratories. Many emerging compounds escape detection by standard targeted assays, leading to underreporting or missed diagnoses. Developing a high-throughput, multiplexed quantitative method enables comprehensive screening and accurate monitoring of patient or forensic samples within a single run.

Objectives and Study Overview

This study aimed to establish and validate a single-assay method for the quantification of 78 drugs of abuse in human whole blood. The method was designed to combine broad compound coverage with robust sensitivity and linearity suitable for clinical research and forensic applications. Key performance metrics included limits of quantification, calibration linearity, and intra-/inter-day precision.

Methodology and Instrumentation

Sample Preparation:

• Negative human whole blood calibrators and controls spiked with 78 analytes and isotope-labeled internal standards across nine calibration levels (0.02–200 ng/mL).
• Protein precipitation with acetonitrile containing internal standards, followed by centrifugation, evaporation, and reconstitution in 90:10 water/methanol.

Liquid Chromatography:

• UltiMate 3000 RSLC equipped with Accucore Phenyl Hexyl column (100 × 2.1 mm, 2.6 μm) at 40 °C.
• Gradient elution with 0.1% formic acid in water and acetonitrile over a 17-minute runtime at 0.5 mL/min.

Mass Spectrometry:

• Q Exactive Focus hybrid quadrupole-Orbitrap with heated electrospray ionization, positive/negative switching.
• Full-scan acquisition m/z 100–600 at 35,000 resolution (FWHM @ m/z 200), 5 ppm mass accuracy window.

Main Results and Discussion

The method demonstrated linear calibration ranges for all analytes, with limits of quantification between 0.02 and 20 ng/mL depending on compound chemistry. Representative chromatograms for low-level calibrators showed sharp, baseline-resolved peaks for baclofen (1 ng/mL), EDDP (0.2 ng/mL), and norbuprenorphine (1 ng/mL). Three-day validation exhibited biases within ±20% at LLOQ and ±15% for higher levels, meeting accepted bioanalytical criteria.

Benefits and Practical Applications

This workflow streamlines routine clinical or forensic testing by combining broad analyte coverage with simple offline preparation. A single injection yields quantitative results for 78 drugs, reducing analysis time and resource consumption. The high resolution and accurate mass detection ensure selectivity against interferences, supporting confident identification and quantification in complex matrices.

Future Trends and Applications

Advances in high-resolution mass spectrometry and data processing will enable even larger panels, including novel psychoactive substances. Integration with automated sample handling and real-time data review can further accelerate turnaround. Expanding panels to other biological fluids and employing retrospective data mining will enhance monitoring of emerging drug use patterns.

Conclusions

A robust LC-HRAM Orbitrap method for quantifying 78 drugs of abuse in whole blood has been successfully developed and validated. The approach meets clinical research requirements for sensitivity, linearity, and throughput while offering extensive compound coverage in a single assay.

References

Thermo Fisher Scientific. Technical Note 73168. Quantification of 78 drugs of abuse in human blood by liquid chromatography–HRAM Orbitrap mass spectrometry for clinical research.