Robustness in Regulated Bioanalysis: A 30,000 Injection Study of Naltrexone in Human Plasma using the Xevo™ TQ Absolute XR Mass Spectrometer

Importance of the Topic

Reliable quantification of pharmaceuticals and metabolites at trace levels in complex biological matrices underpins critical decisions in drug development, therapeutic monitoring, and regulatory compliance. High-throughput bioanalytical laboratories require robust workflows that sustain long-term stability, minimize downtime, and deliver consistent data quality at sub-ng/mL concentrations. Addressing challenges such as matrix effects, ion suppression, and instrument contamination is essential to maintain productivity and confidence in pharmacokinetic and safety studies.

Study Objectives and Overview

This study evaluated the long-term robustness and quantitative performance of the Xevo TQ Absolute XR Mass Spectrometer for the analysis of naltrexone and its active metabolite 6β-naltrexol in human plasma. Over 30,000 sequential injections were performed to simulate a high-throughput regulated bioanalysis workflow. Key performance indicators—including sensitivity, precision, accuracy, chromatographic integrity, and carryover—were monitored to confirm instrument stability and method compliance with FDA M10 bioanalytical validation criteria.

Methodology

Samples were prepared by protein precipitation in 96-well format using human plasma spiked with certified standards of naltrexone, 6β-naltrexol, and deuterated internal standards. Calibration curves ranged from 0.2 to 100 ng/mL, with quality control (QC) levels at 0.2, 0.6, 40, 80, and 100 ng/mL. Each 96-well plate contained calibration standards, QC replicates, system suitability, blanks, and carryover checks. A total of 64 plates (30,720 injections) were analyzed, with each plate injected five times before replacement. Weekly source cleaning was performed without venting the MS vacuum to mimic routine laboratory maintenance.

Instrumentation

The analytical setup comprised an ACQUITY Premier LC System with a reversed-phase column (column temperature 60 °C, flow rate 0.8 mL/min, 2.1-minute gradient) coupled to the Xevo TQ Absolute XR Triple Quadrupole Mass Spectrometer operating in positive electrospray ionization. The instrument featured the StepWave XR slotted bandpass ion guide to prevent high m/z ion contamination of the first quadrupole. Data acquisition and review were conducted in waters_connect for Quantitation Software using a review-by-exception approach.

Main Results and Discussion

Sustained sensitivity and quantitative integrity were observed over 55 days of continuous acquisition. More than 21 mL of plasma was injected without instrument downtime. All 320 individual plate runs met FDA M10 acceptance criteria for calibration linearity, QC accuracy (±15%, ±20% at LLOQ), precision, and absence of carryover. At the 0.2 ng/mL LLOQ, replicate peak areas at early (~1,000), midpoint (~15,000), and final (~30,000) injections exhibited %RSD below 3.0%. Calculated concentrations remained within ±10% of nominal values for naltrexone and 6β-naltrexol, with over 99.6% of LLOQ QC data points meeting ±20% criteria. Precision across all QC levels stayed below 3.5% RSD in most cases.

Benefits and Practical Applications

• Extended uptime with no unplanned maintenance supports uninterrupted high-throughput bioanalysis.
• Regulatory compliance at sub-ng/mL concentrations ensures reliable pharmacokinetic and toxicokinetic data.
• Streamlined data review in waters_connect reduces analyst workload and accelerates result verification.

Future Trends and Opportunities

Emerging developments in ion‐guide design, automation of source cleaning, and advanced software algorithms for automated flagging will further enhance instrument robustness and data quality. Integration of predictive maintenance and machine-learning-driven quality control can optimize long-term performance and reduce operational costs. Expanding workflows to multi-analyte panels and novel biomarkers will benefit from proven stability in demanding high-throughput settings.

Conclusion

The Xevo TQ Absolute XR Mass Spectrometer equipped with StepWave XR demonstrated exceptional robustness across over 30,000 injections in a regulated bioanalysis context. Consistent sensitivity, precision, and accuracy at sub-ng/mL levels confirm its suitability for high-throughput discovery and regulated laboratories, delivering reliable performance with minimal downtime.

References

1. Shen PT, Liu SC, Huang MC, Liu YL. Development of a liquid chromatography-tandem mass spectrometry method for the analysis of plasma naltrexone and its active metabolite in patients with AUD. Eur J Mass Spectrom (Chichester). 2025;14690667251384047.
2. Timmerman P, McDougall S, et al. European Bioanalysis Forum recommendation on context-of-use-driven validation for chromatographic assays in light of ICH M10. Bioanalysis. 2025;1–10.