Quantification of HIV Antiretroviral Drugs in Human Plasma by Matrix Assisted Laser Desorption Ionisation Time-of-Flight Mass Spectrometry

Significance of the topic

The ability to quantify small-molecule drugs in human plasma rapidly and with minimal sample handling is important for therapeutic drug monitoring, pharmacokinetic studies and high-throughput clinical research. MALDI-TOF MS is widely used for sensitive and robust mass measurement but has historically been considered unsuitable for quantitative bioanalysis because of heterogeneous analyte/matrix crystallisation and spot-to-spot ion intensity variability. Demonstrations that adopt internal standard normalisation and validation against regulatory guidance can broaden MALDI-TOF MS utility for targeted drug quantification, offering simplified workflows versus LC–MS/MS and enabling higher sample throughput.

Objectives and study overview

This work presents a proof-of-principle assay for absolute quantification of the HIV protease inhibitor lopinavir (LPV) in human plasma using a Shimadzu MALDI-8030 EasyCare MALDI-TOF instrument, with ritonavir (RTV) as the internal standard (I.S.). The assay was developed to follow FDA bioanalytical validation criteria and to evaluate linearity, precision, accuracy and lower limit of quantification (LLOQ) when using a MALDI-based workflow without chromatographic separation.

Sample preparation and methodology

The procedure emphasises minimal and rapid sample processing:

• Start with 10 µL human plasma; spike with LPV calibrators and a fixed RTV I.S. (final RTV = 500 nM).
• Add 70 µL methanol (protein precipitation and dilution to 100 µL total); extract 1 hour at 4 °C and centrifuge 5 min at 15,000 rpm.
• Mix an aliquot of supernatant 1:1 with CHCA MALDI matrix (5 mg/mL in 50:50 acetonitrile/water) containing 50 mM sodium trifluoroacetate (NaTFA) as cationising agent.
• Spot 1 µL of the sample/matrix premix onto the MALDI target and acquire spectra.

Calibration design and validation:

• Seven calibrators spanning 75 nM–1.5 µM (75 nM set as LLOQ).
• Five replicates per calibrator; quality controls at low (225 nM), mid (600 nM) and high (1.2 µM) levels.
• A blind plasma sample containing 400 nM LPV was used to test accuracy.

Ionisation strategy and data handling:

• Positive-ion MALDI analysis; drugs detected primarily as sodiated species [M + Na]+ (LPV calculated m/z 651.352; RTV calculated m/z 743.302).
• Lock-mass correction applied using the RTV m/z to improve mass accuracy.
• Peak-height ratios (LPV/RTV) averaged across replicates were used to construct the calibration curve.

Used instrumentation

The experimental work used:

• Shimadzu MALDI-8030 EasyCare benchtop MALDI-TOF mass spectrometer operated in positive ion mode (acquisition parameters: m/z range ~100–1000, multiple shots per profile; lock-mass correction applied).
• α-Cyano-4-hydroxycinnamic acid (CHCA) as MALDI matrix, prepared at 5 mg/mL in 50:50 acetonitrile/water with 50 mM NaTFA as cationising agent.
• Analytical standards of lopinavir and ritonavir, human plasma from a commercial supplier, and routine centrifugation equipment for protein precipitation/extraction.

Main results and discussion

Key analytical findings:

• The LPV/RTV peak-height ratio produced a calibration curve with excellent linearity (coefficient of determination R² = 0.9987) across the tested range.
• The assay achieved an LLOQ of 75 nM for LPV.
• Precision and accuracy for calibrators and QCs met FDA bioanalytical method validation criteria (precision CV ≤15% for all levels except LLOQ where ≤20%; accuracy bias within ±15% for all levels except LLOQ where ±20% was acceptable).
• The blind sample containing 400 nM LPV was quantified as 372 nM, demonstrating acceptable accuracy within guideline limits.

Interpretation:

The use of an exogenous internal standard (RTV) to normalise signal variability effectively compensated for MALDI spot heterogeneity and produced reliable quantitative results. Detection as sodiated adducts was promoted by NaTFA addition, improving reproducible ion formation for both analyte and I.S. The simple protein-precipitation extraction and absence of chromatographic separation substantially shorten preparation and analysis time compared with typical LC–MS/MS workflows, enabling higher throughput while still satisfying regulatory validation endpoints.

Benefits and practical applications

Practical advantages demonstrated by the study include:

• Minimal sample volume requirement (10 µL plasma) and straightforward sample preparation compatible with batch processing.
• Rapid data acquisition without chromatographic separation, increasing throughput for pharmacokinetic screening, clinical research and preclinical studies.
• Regulatory-aligned validation (FDA guidance) showing that MALDI-TOF can meet precision and accuracy benchmarks when an appropriate internal standard strategy is used.

Potential application areas:

• Therapeutic drug monitoring where speed and sample throughput are priorities.
• High-throughput pharmacokinetic screening in early drug development or clinical studies.
• Targeted small-molecule assays in laboratories where MALDI-TOF instruments are already in routine use.

Future trends and potential uses

Opportunities and developments to broaden MALDI-based quantification include:

• Use of isotopically labelled internal standards to more closely mimic analyte behaviour and further reduce matrix effects.
• Automation of spotting and sample handling to improve reproducibility and throughput for large studies.
• Method transfer and inter-laboratory validation to support broader regulatory acceptance for clinical applications.
• Extension to additional drug classes, metabolites or multiplexed assays where simultaneous measurement of several analytes is desirable.
• Integration with imaging or ambient ionisation approaches for spatially resolved or direct-from-sample analyses when appropriate.

Remaining challenges include managing ion suppression across diverse clinical matrices, ensuring batch-to-batch spot uniformity, and demonstrating robustness across different instrument platforms and operators.

Conclusion

This study provides a practical proof-of-principle that MALDI-TOF MS, when combined with an appropriate internal standard strategy and a simple protein-precipitation workflow, can produce validated quantitative results for small-molecule drugs in human plasma. The approach delivered excellent linearity, met FDA validation criteria, required minimal sample handling and provided higher throughput potential compared with LC–MS/MS, supporting further exploration and method refinement for targeted bioanalysis applications.

References

1. van Kampen JJA, Reedijk ML, Burgers PC, Dekker LJM, Hartwig NG, et al. Ultra-Fast Analysis of Plasma and Intracellular Levels of HIV Protease Inhibitors in Children: A Clinical Application of MALDI Mass Spectrometry. PLoS ONE 2010;5(7):e11409.
2. Guidance for Industry: Bioanalytical Method Validation. U.S. Food and Drug Administration, 2018.