Extraction and Analysis of Orthocetamol, Metacetamol and Paracetamol in Human Plasma Using Oasis™ PRiME HLB and Waters ACQUITY™ Biphenyl RP Column with MaxPeak™ Technology

Importance of the topic

The ability to separate and quantify positional isomers of acetaminophen (orthocetamol, metacetamol, paracetamol) in human plasma is important for forensic, clinical and bioanalytical laboratories. Positional isomers share identical nominal mass and often very similar physicochemical properties, making chromatographic resolution essential to avoid misidentification or inaccurate quantitation when using mass-selective detectors. Robust, high-throughput sample cleanup that minimizes matrix effects and protects column lifetime is also critical in routine bioanalysis.

Objectives and overview of the study

This application study aimed to develop a concise workflow that:

• achieves baseline chromatographic separation of three positional isomers of acetaminophen;
• provides a fast, reproducible sample cleanup from pooled human plasma using a simple SPE protocol;
• yields high recovery and low matrix effects compatible with LC-MS detection.

The study evaluated neat standards and plasma samples spiked with the three isomers, combining Oasis PRiME HLB SPE with a Waters ACQUITY BEH Biphenyl RP column employing MaxPeak surface technology and SIR mass detection at m/z 152.

Methodology

Sample preparation:

• A single stock containing 1 mg/mL of each isomer was prepared; plasma was pre-spiked to 80 µg/mL then processed and diluted to a final analyzed concentration of 20 µg/mL per analyte.
• Samples (pre-spike, post-spike, blank/non-matrix) were acidified 1:1 with 4% phosphoric acid before SPE to ensure analytes were neutral and retained by the hydrophobic sorbent.
• Extraction employed an Oasis PRiME HLB 96-well plate using a semi-automated positive-pressure manifold and a 2-step protocol (load and elute) designed to limit preparation time while removing phospholipids and polar interferences.
• Final extracts were diluted with water prior to injection to avoid chromatographic disturbance from high organic eluent.

Chromatography and detection:

• Column: Waters ACQUITY BEH Biphenyl with MaxPeak Technology, 2.1 x 50 mm, 1.7 µm; column temperature 30 °C.
• Mobile phases: A – water; B – methanol; additive maintained as 2% formic acid (held at constant 5% D to maintain additive concentration).
• Gradient: 5% B ramped to 95% B over 5.00 min, held 1.00 min, return and re-equilibrate for total run ~8.00 min; flow 0.4 mL/min; injection volume 1.0 µL; sample temp 10 °C.
• Detection: ACQUITY QDa mass detector operated in positive ESI, SIR at 152 m/z, capillary 1.5 kV, cone 15 V.

Used instrumentation

• ACQUITY UPLC H-Class Plus system with CM and CM Aux modules
• ACQUITY PDA detector
• ACQUITY QDa mass detector (single ion recording at 152 m/z)
• ACQUITY BEH Biphenyl RP Column with MaxPeak Technology, 2.1 x 50 mm, 1.7 µm
• Oasis PRiME HLB 96-well plate (30 mg sorbent per well) and semi-automated positive-pressure manifold
• Empower Chromatography Data System for data handling

Key results and discussion

The BEH Biphenyl stationary phase with MaxPeak surface treatment delivered baseline resolution of paracetamol and its ortho- and meta- positional isomers, producing sharp symmetrical peaks suitable for isomer discrimination by retention time in combination with SIR at m/z 152. Blank plasma processed through the SPE showed no endogenous interference at the analyte retention times, supporting accurate spike-level assessment.

Extraction performance: replicate analyses (n=4) showed recoveries greater than 90% for all three isomers and minimal matrix effects, indicating that the Oasis PRiME HLB 2-step protocol effectively removed phospholipids and common matrix interferences while retaining the analytes. The short gradient and small injection volume produced an 8-minute total cycle time compatible with higher-throughput bioanalytical workflows.

The combination of a biphenyl ligand (improved isomer selectivity), BEH particle chemistry (batch-to-batch reproducibility and pH stability) and MaxPeak hardware surface technology (reduced analyte–metal interactions) contributed to method robustness and reproducibility across runs.

Benefits and practical applications

• Reliable baseline separation of isobaric positional isomers allows positive identification by retention time and mass, essential when m/z alone is insufficient.
• High extraction recoveries (>90%) and low matrix effects support accurate quantitation in plasma matrices.
• Short run time (8 min) and 96-well SPE compatibility enable moderate-to-high throughput for routine bioanalytical testing, toxicology screens, or pharmacokinetic sample analysis.
• Reduced column fouling and extended column lifetime due to efficient phospholipid removal and MaxPeak surface technology.

Future trends and potential applications

Continued advances that can extend and generalize this workflow include:

• Application to other sets of positional or structural isomers in clinical and forensic matrices, leveraging biphenyl selectivity;
• Integration with tandem MS/MS or high-resolution MS workflows to provide orthogonal confirmation when required;
• Automation of the Oasis PRiME HLB SPE workflow for fully unattended sample processing and scaling to larger cohorts;
• Exploration of ultra-short biphenyl columns and faster gradients for increased sample throughput while preserving isomer resolution;
• Use of surface-treated hardware and particle technologies to further mitigate adsorption losses for polar or labile analytes.

Conclusion

The combined Oasis PRiME HLB 2-step SPE protocol and ACQUITY BEH Biphenyl column with MaxPeak Technology provide a robust, rapid and high-recovery workflow for resolving and quantifying orthocetamol, metacetamol and paracetamol in human plasma. Baseline chromatographic separation plus sensitive SIR detection enabled confident discrimination of these isobaric analytes with minimal matrix impact, making the method suitable for routine bioanalytical and forensic applications.

References

The summary is based on an application note reporting: use of Oasis PRiME HLB SPE 2-step protocol for rapid phospholipid removal; validation of BEH particle and MaxPeak surface technologies for reproducibility and mitigation of analyte–metal interactions; and literature demonstrating biphenyl phase selectivity for isomer separations. Specific citations and Waters application notes and journal articles were referenced in the original report.