Novel Extraction Techniques with ACQUITY UPLC with 2D Technology: Part II Ketamine and Xylazine in Bone Matrix

Importance of the topic

The quantitative analysis of ketamine and xylazine in bone matrices addresses a growing need in forensic and toxicological investigations. Conventional specimens such as blood or urine may degrade or fail to retain trace compounds over time. Bone, thanks to its durability, offers a stable reservoir for detecting anesthetic, veterinary, or illicit agents long after an incident. Optimizing extraction and chromatographic methods for these challenging solid matrices improves sensitivity, reduces sample-prep time, and broadens the scope of post-mortem and forensic testing.

Objectives and overview of the study

This application note evaluates novel extraction strategies—captive and passive solid-phase extraction—coupled with two-dimensional ultrahigh-performance liquid chromatography (2D UPLC) and tandem mass spectrometry for the determination of ketamine and xylazine in rat bone matrix. The primary goals are:

• To develop a rapid, automated sample-prep protocol capable of achieving parts-per-trillion detection limits.
• To compare captive versus passive extraction workflows for matrix cleanup and analyte recovery.
• To demonstrate the utility of 2D chromatography with at-column dilution to streamline method development and improve selectivity.

Methodology

Bone samples from xylazine/ketamine-treated rats were homogenized and centrifuged. The supernatant was diluted for two distinct extraction approaches:

• Captive extraction: Target analytes are retained on a reversed-phase sorbent during loading, washed to remove interferences, and eluted under optimized pH and solvent conditions.
• Passive extraction: A blended polymer-based SPE sorbent generates a controlled breakthrough of analytes during loading, followed by elution in a single step without discrete wash phases.

An automated 6×6 grid optimization tested combinations of sorbent chemistries (C8, C18, HLB), loading pH (3, 7, 10), and elution solvents (acetonitrile or methanol with 0.5% formic acid). Chromatographic separation employed a trap-and-elute configuration on ACQUITY UPLC BEH C18 (1.7 µm) and Xevo TQ-S MS detection in positive electrospray MRM mode.

Used instrumentation

• Waters ACQUITY UPLC System with 2D Technology (trap & elute, at-column dilution)
• Xevo TQ-S triple quadrupole mass spectrometer with ESI source
• MassLynx v4.1 data acquisition software
• Oasis HLB and Oasis PRiME HLB SPE cartridges
• XBridge and ACQUITY UPLC BEH C18 columns

Main results and discussion

Initial MRM optimization achieved two transitions each for quantification and confirmation of ketamine (m/z 238.2 → 125.0, 222.2) and xylazine (m/z 221.2 → 129.1, 164.0). The 6×6 screening identified neutral-pH loading onto Oasis HLB with low-pH acetonitrile elution as the most cost-effective method. Captive extraction profiling showed:

• Ketamine and xylazine elute sharply at 20–30% acetonitrile (pH 3) and 30–50% acetonitrile (pH 10), reflecting pKa-dependent retention behavior.
• An optimized wash step (20% methanol, pH 10) and elution (40% methanol, pH 3) reduced matrix interferences and maintained recoveries above 90%.

Passive extraction achieved similar recoveries (>87%) using a single 50/50 water/MeOH (pH 3) breakthrough and elution, simplifying workflow and avoiding solvent exchanges. Solid-liquid extraction comparison on rat bone homogenate found >90% recovery for both analytes with methanol and acetonitrile at pH 10.

Benefits and practical applications of the method

The combined 2D UPLC–MS/MS platform with optimized SPE enables:

• A rapid 30-minute end-to-end protocol without evaporation or reconstitution.
• Trace-level detection at parts-per-trillion with minimal sample handling.
• Robust performance across complex solid matrices, expanding forensic and veterinary residue testing.

Future trends and potential applications

Continued advancements in multidimensional chromatography and mixed-mode sorbents will further reduce sample-prep steps and improve throughput. Potential extensions include:

• Screening additional veterinary or illicit compounds in bone, hair, and entomological samples.
• Integration with high-resolution MS for non-targeted screening in complex post-mortem matrices.
• Automation of passive SPE formats in 96-well plates for high-throughput forensic workflows.

Conclusion

This study demonstrates that both captive and passive SPE protocols, when coupled with ACQUITY UPLC 2D Technology and Xevo TQ-S MS, deliver high recoveries and trace-level quantitation of ketamine and xylazine in bone matrix. Method development is accelerated by automated grid optimization, and the final 30-minute workflow offers clear advantages for forensic toxicology laboratories.

References

• 1. Mella M, Schweitzer B, Mallet CR, Moore T, Botch-Jones S. Novel Extraction Techniques with ACQUITY UPLC 2D: Ketamine and Xylazine in Bone Matrix. J Anal Toxicol. 2018;42(4):265–275.
• 2. Mallet CR, Botch-Jones S. Automated 2D UPLC Workflow Development for Multi-Residue Screening. J Anal Toxicol. 2016;40(1):1–11.
• 3. Mallet CR. Multidimensional Chromatography Compendium: Trap & Elute vs. At-Column Dilution. Waters Corp. Application Note 720005339EN; 2015.
• 4. Mallet CR. Analysis of Pharmaceuticals and Pesticides in Water by ACQUITY UPLC 2D Technology. Waters Corp. Application Note 720005167EN; 2014.