Novel Extraction Techniques Using ACQUITY UPLC with 2D Technology: Part IV – First Time Users

Importance of the Topic

Rapid and sensitive detection of emerging contaminants such as synthetic cannabinoids and macrolide antibiotics in biological fluids is critical for forensic toxicology, clinical diagnostics, and environmental monitoring. Multidimensional liquid chromatography combined with tandem mass spectrometry expands separation capacity and reduces extensive sample preparation steps, enabling trace-level quantitation in complex matrices.

Objectives and Study Overview

This work aimed to develop and validate a streamlined two-dimensional UPLC-MS/MS workflow for first-time users to screen and quantify multiple drug classes in urine. A five-day training program guided inexperienced analysts through method development, chromatographic optimization, and sample preparation using ACQUITY UPLC with 2D Technology.

Used Instrumentation

• ACQUITY UPLC with 2D Technology
• ACQUITY UPLC BEH C18 Columns
• XBridge BEH C18 Columns
• Oasis HLB Direct Connect HP Columns
• Xevo TQ-S Triple Quadrupole Mass Spectrometer
• MassLynx Mass Spectrometry Software v4.1

Methodology

• Optimized multiple reaction monitoring transitions at pH 3, 7, and 10 by direct infusion to select the most intense precursor and product ion pairs.
• Employed a 6×6 method development grid varying trap chemistry (C8, C18, HLB), loading pH, and elution solvent/ pH. Selected optimal methods for cannabinoids (C18 trap, pH 3 loading, acetonitrile elution at pH 10) and macrolides (HLB trap, pH 3 loading, acetonitrile elution at pH 3).
• Performed solid-phase extraction on Oasis HLB cartridges with four steps (condition, load, wash with 5% MeOH, elute) and eliminated evaporation and reconstitution by direct injection into the 2D-LC system.
• Constructed seven-point calibration curves (0.1–10 ng/mL) for each analyte in methanol and confirmed linearity (r²>0.995) across three orders of magnitude.

Main Results and Discussion

• Limits of detection reached 0.1 ng/mL (sub-ppb) for all targets; some analytes indicated potential detection at 0.01 ng/mL.
• SPE recoveries in aqueous standards exceeded 95% under optimal elution pH; urine matrix recoveries remained above 75%, demonstrating effective cleanup despite mild wash conditions.
• Chromatograms exhibited sharp Gaussian peak shapes, high signal-to-noise ratios, and no significant interferences at target retention times.
• Elimination of evaporation and reconstitution reduced sample preparation time to under 45 minutes per batch without compromising sensitivity or reproducibility.

Benefits and Practical Applications

• Fast extraction workflows and high throughput support routine screening and confirmatory analyses in forensic, clinical, and pharmaceutical laboratories.
• Trace-level quantitation down to parts-per-trillion facilitates monitoring of low-abundance analytes and emerging compounds.
• Enhanced separation power of 2D chromatography reduces coelution problems and improves method robustness.

Future Trends and Potential Applications

Emerging developments include automated method-development algorithms for multidimensional chromatography, integration with high-resolution mass spectrometry, expansion to diverse compound classes and matrices, and implementation of artificial intelligence-driven data processing to further improve sensitivity, reduce analysis time, and support real-time decision making.

Conclusion

The combination of ACQUITY UPLC with 2D Technology and simplified SPE enables first-time users to achieve rapid, sensitive, and reproducible analysis of synthetic cannabinoids and macrolide antibiotics in urine. The approach delivers high separation capacity, trace-level detection, and significant reductions in sample preparation time, making it a valuable workflow for modern analytical laboratories.

References

• Benvenuto K; Botch-Jones S; Mallet C. Analysis of Synthetic Cannabinoids in Urine, Plasma, and Edibles Using Multidimensional UPLC Technology. Application Note – Waters Corporation, 720006015EN, 2017.
• Mallet C; Botch-Jones S. Illicit Drug Analysis in Urine Using 2-D LC-MS/MS for Forensic Toxicology. Application Note – Waters Corporation, 720005533EN, 2017.
• Mallet C; Liu Y; Novack T. Optimized Workflow for API Process Impurities Using 2D LC-MS/MS. Application Note – Waters Corporation, 720005573EN, 2016.
• Mella M; Schweitzer B; Botch-Jones S; Mallet C. Analysis of Ketamine and Xylazine in Rat Tissues Using the ACQUITY UPLC with 2D Technology. Application Note – Waters Corporation, 720005810EN, 2016.
• Mella M; Schweitzer B; Botch-Jones S; Mallet C; Kemp P; Cliburn K; Canfield S. Rapid Analysis of Pharmaceuticals in Human Tissues Using the ACQUITY UPLC with 2D Technology. Application Note – Waters Corporation, 720005896EN, 2017.
• Mella M; Schweitzer B; Botch-Jones S; Mallet C. 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. Application Note – Waters Corporation, 720005847EN, 2016.