COMPARISON OF DATA-INDEPENDENT AND DATA- DEPENDENT TECHNIQUES FOR FORENSIC TOXICOLOGY SCREENING ANALYSIS

Significance of the topic

In forensic toxicology, the growing number and diversity of drugs present significant analytical challenges. High-resolution mass spectrometry has become a key tool for comprehensive screening, offering enhanced specificity and sensitivity for the detection of both known and emerging substances.

Objectives and study overview

This work evaluated and compared two mass spectrometric screening strategies on a UPLC-QTof system: data-independent acquisition (DIA) using MSE and data-dependent acquisition (DDA) with two trigger-based variants. The comparison addressed method setup, data complexity, processing time, and screening performance in controlled and real urine samples.

Methodology

Samples included blank urine spiked with a 25-analyte system suitability mixture (50 ng/mL) and twenty anonymized authentic urine specimens previously characterized by multiple techniques. All samples were diluted fivefold in aqueous mobile phase prior to analysis. Chromatography was performed with a 15-minute gradient on a 2.1×150 mm C18 column at 50 °C, using 5 mM ammonium formate (pH 3.0) and acetonitrile with 0.1 % formic acid. The mass spectrometer operated in positive electrospray mode over m/z 50–1000.

Used instrumentation

• ACQUITY UPLC I-Class system
• Xevo G3 QTof mass spectrometer
• waters_connect UNIFI informatics platform

Main results and discussion

In screening 146 reference analytes, the MSE (DIA) approach detected 94 %, while DDA-1 and DDA-2 achieved 86 % and 82 % respectively. MSE data processing required approximately 3 minutes per sample versus 12 minutes for each DDA workflow. MSE showed fewer false negatives, particularly for low-abundance compounds, as it fragments all ions without relying on intensity thresholds. DDA methods missed analytes due to trigger conflicts and the absence of full-scan data during MS/MS acquisition. The unrestricted nature of MSE supports retrospective mining of the complete dataset.

Benefits and practical applications

• Simplified method setup without optimization of trigger thresholds
• Comprehensive and unbiased fragmentation of all ions
• Faster data processing supporting high-throughput screening
• Reduced false negatives and enhanced detection of low-level analytes
• Complete data acquisition enabling retrospective analysis

Future trends and possibilities

Advances in data mining and machine learning are expected to enhance automated identification from DIA datasets. Expansion of spectral libraries and integration with novel separation techniques will broaden applicability across biological matrices. Real-time or near-real-time screening workflows may emerge, leveraging unrestricted acquisition modes.

Conclusion

The MSE-based DIA method outperformed traditional DDA strategies in ease of use, processing speed, and detection efficiency. Its unbiased acquisition offers a robust platform for forensic toxicology screening, reducing false negatives and supporting comprehensive forensic investigations.

References

1. Wood M. Utility of MSE for Toxicological Screening. Waters Application Note, 720005198, Mar 2022.
2. Wood M, Mistry NS. Quality Over Quantity—It’s Not Always a Numbers Game. Waters White Paper, 720007520, Mar 2022.