Quantitative LC/MS/MS Analysis of Drugs in Human Serum With Agilent Captiva EMR–Lipid Cleanup

Importance of the topic

Efficient quantification of drug compounds in human serum is essential for clinical research, pharmacokinetics, therapeutic drug monitoring, and toxicology studies. Removing lipids and other endogenous interferences preserves instrument performance, minimizes ion suppression, and supports reliable, high-throughput bioanalysis.

Objectives and study overview

This work compares a combined protein precipitation (PPT) and Agilent Captiva EMR–Lipid cleanup protocol against PPT alone for nine representative drugs in human serum. The main goals are:

• To establish a calibration range of 0.5–200 ng/mL with linearity (R² > 0.99).
• To assess accuracy and precision at LLOQ, low, mid, high, and HLOQ levels.
• To evaluate matrix effects and ion suppression profiles.
• To verify method selectivity, carryover, and batch-to-batch consistency of the sorbent.

Methodology and sample preparation

Serum aliquots (100 µL) were spiked with analytes and internal standards, followed by in-well PPT using 1% formic acid in acetonitrile. The supernatant was transferred to a Captiva EMR–Lipid 96-well plate for pass-through cleanup via size exclusion and hydrophobic interactions. After vacuum elution, samples were evaporated at 40 °C and reconstituted in 10:90 acetonitrile/5 mM ammonium acetate buffer.

Used Instrumentation

• Agilent 1290 Infinity II UHPLC with Poroshell 120 EC-C18 column (150 × 2.1 mm, 2.7 µm).
• Agilent 6490 Triple Quadrupole LC/MS with Jet Stream iFunnel ESI source.
• Agilent Captiva EMR–Lipid 96-well cleanup plates.
• CentriVap concentrator and standard vacuum manifold components.

Main results and discussion

The combined PPT and EMR–Lipid method achieved excellent calibration linearity (R² > 0.99) and met bioanalytical criteria: accuracy within ±20% at LLOQ and ±15% at other levels, and RSD < 15%. Post-column infusion demonstrated that PPT-only samples suffered significant ion suppression in two major retention windows due to phospholipids, whereas Captiva EMR–Lipid cleanup eliminated these suppression zones. Sorbent reproducibility was confirmed across three lots, each delivering >99% phospholipid removal and consistent analyte recovery.

Benefits and practical applications

• Enhanced assay robustness by reducing matrix interferences.
• High-throughput 96-well format compatible with large sample loads.
• Possibility to use structural analog internal standards when isotopic labels are not available.
• Streamlined workflow for clinical, pharmaceutical, and routine QA/QC laboratories.

Future trends and potential uses

Future developments may focus on extending EMR–Lipid cleanup to multispecies analyte panels in complex foods or environmental matrices, integrating the workflow with automated liquid handlers, and coupling with high-resolution mass spectrometry for comprehensive metabolite profiling.

Conclusion

The PPT plus Agilent Captiva EMR–Lipid protocol offers a robust, efficient solution for quantitative LC/MS/MS analysis of drugs in serum, delivering superior linearity, accuracy, precision, and minimal matrix effects compared to PPT alone.

Reference

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