LC/MS/MS High Sensitivity Bioanalytical Method: Entecavir in Human Plasma

Significance of the Topic

The quantitative measurement of entecavir in human plasma is critical for therapeutic drug monitoring, pharmacokinetic profiling and bioequivalence studies in hepatitis B treatment. Highly sensitive and robust bioanalytical methods enable accurate dosing, support regulatory requirements and streamline pharmaceutical development.

Objectives and Study Overview

This work aimed to develop a rapid, high-sensitivity LC/MS/MS assay for entecavir in human plasma using minimal sample preparation. Key goals included:

• Achieve a lower limit of quantification of 10 pg/mL in plasma
• Eliminate solid-phase or liquid-liquid extraction by using protein precipitation alone
• Demonstrate precision, accuracy and linearity across a wide concentration range
• Ensure high throughput and low operating cost

Methodology and Instrumentation

Plasma samples spiked with entecavir were deproteinated by adding cooled acetonitrile:methanol (1:1) followed by vortexing, centrifugation and filtration. A 1 µL aliquot was injected onto a Kinetex Phenyl-Hexyl column (150 × 2.1 mm, 2.6 µm) using a 10-minute gradient of 5 mM ammonium formate with 0.01% formic acid in water and methanol at 0.3 mL/min. Detection was performed in positive ESI mode with multiple reaction monitoring transitions m/z 278→152 for quantification and m/z 278→135 for confirmation. Calibration standards ranged from 10 to 10 000 pg/mL.

Used Instrumentation

• Shimadzu Nexera X2 UHPLC system
• Shimadzu LCMS-8060 triple quadrupole mass spectrometer with heated ESI
• Kinetex 2.6 µm Phenyl-Hexyl column, 100 Å

Main Results and Discussion

The assay exhibited a linear response (R² > 0.999) between 10 and 10 000 pg/mL. The LLOQ of 10 pg/mL in plasma (40 pg/mL pre-precipitation) provided a signal-to-noise ratio near 10. Within-run precision and accuracy at LLOQ and QC levels met acceptance criteria (≤15% RSD, ±15% accuracy; ≤20% at LLOQ). Extraction recovery averaged 97–114% and matrix effects showed moderate ion enhancement (~167% mean), compensated through calibration. Chromatographic peaks were sharp at ~4.4 min retention time and method reproducibility demonstrated by overlay of consecutive injections.

Benefits and Practical Applications

This streamlined bioanalytical protocol offers several advantages:

• Minimal sample preparation with protein precipitation only
• High sensitivity suitable for low-level pharmacokinetic studies
• Rapid analysis cycle for high throughput
• Reduced costs by avoiding SPE or LLE consumables

Future Trends and Opportunities

Advances in microflow LC, automated on-line sample cleanup and high-resolution mass spectrometry may further enhance sensitivity and specificity. Integration with robotic liquid handling and data processing pipelines will support large-scale clinical studies and personalized medicine applications. Emerging ionization technologies and column chemistries can expand the assay to related nucleoside analogs.

Conclusion

A robust and high-sensitivity LC/MS/MS method for entecavir quantitation in human plasma was established using protein precipitation and a Shimadzu LCMS-8060 system. The assay meets regulatory bioanalytical criteria, supports pharmacokinetic research and offers cost-effective, high throughput performance.

References

1. Duxi Zhang et al. A sensitive method for the determination of entecavir at pictogram per milliliter level in human plasma by solid phase extraction and high-pH LC-MS/MS. J. Pharm. Biomed. Anal. 49 (2009) 1027–1033.
2. Balasekhara Reddy Challa et al. LC-ESI-MS/MS method for the quantification of entecavir in human plasma and its application to a bioequivalence study. J. Chromatogr. B 879 (2011) 769–777.
3. Suraj Sythana et al. Determination of entecavir in human plasma by LC-MS/MS and method validation. Int. J. PharmTech Res. 4(4) (2012) 1721–1729.