Quantitation

Mass spectrometric detection for quantitation is most commonly performed on triple quadrupole instruments in multiple reaction monitoring (MRM/SRM) mode, providing unmatched selectivity and linear dynamic range. Transitions are selected based on the most abundant, specific product ions; collision energies, cone voltages, and dwell times are optimized to maximize signal-to-noise while preserving sufficient points across the peak. Scheduled MRM further increases duty cycle in multiplexed assays. High-resolution MS (e.g., PRM on Q-TOF) may be leveraged for challenging selectivity scenarios, for metabolite confirmation, or when interferences are suspected.

Calibration, quality control, and validation Calibrators are prepared by spiking reference standards into the same biological matrix to create matrix-matched standard curves, typically spanning from the lower limit of quantitation (LLOQ) through expected Cmax, with 6–8 non-zero levels. Weighted regression (1/x or 1/x^2) is commonly used to reduce heteroscedasticity across the dynamic range. Independent quality control (QC) samples at LLOQ, low, mid, and high levels are interleaved across the batch and bracketed by calibration standards to monitor assay performance and drift.

Method validation follows current bioanalytical guidance (e.g., ICH M10, FDA, EMA) encompassing:

• Selectivity and specificity across at least six independent matrix lots, including hemolyzed and lipemic matrices when relevant.
• Sensitivity with verified LLOQ meeting accuracy (±20% at LLOQ, ±15% otherwise) and precision criteria.
• Accuracy and precision (intra-/inter-run), recovery, and matrix effect evaluation (e.g., post-extraction spike, post-column infusion).
• Carryover assessment and mitigation via wash protocols and chromatographic adjustments.
• Stability studies: bench-top, freeze–thaw, long-term, and post-preparative autosampler stability; whole-blood stability when applicable.
• Dilution integrity for samples above the upper limit of quantitation.
• Incurred sample reanalysis (ISR) to confirm real-world reproducibility.

Pharmacokinetic analysis Concentration–time data derived from LC–MS quantitation underpin PK parameter estimation. Noncompartmental analysis (NCA) yields Cmax, Tmax, AUC0–t and AUC0–∞, terminal half-life (t1/2), apparent volume of distribution (Vd or Vss), clearance (CL), and mean residence time (MRT). For oral dosing, absolute or relative bioavailability (F) can be determined when IV data or appropriate references are available. Compartmental modeling may be applied for mechanistic insights, flip-flop kinetics, target-mediated disposition, or complex absorption.

By uniting rigorous bioanalytical best practices with targeted LC–MS detection and validation, and by leveraging advanced data processing to ensure data integrity and throughput, LC–MS quantitation provides the quantitative backbone for reliable PK and clearance characterization throughout the drug R&D lifecycle.