Highly Sensitive LC-MS/MS Method for Quantification of Barnidipine in Human Plasma

Importance of the Topic

Barnidipine is a novel dihydropyridine calcium channel blocker prescribed for hypertension. Its therapeutic plasma levels are at picogram-per-milliliter concentrations, demanding a highly sensitive analytical assay. Accurate quantification of barnidipine supports pharmacokinetic profiling, dose optimization and safety monitoring in clinical research.

Objectives and Study Overview

The primary aim was to develop and partially validate a liquid chromatography-tandem mass spectrometry method capable of quantifying barnidipine at picogram levels in human plasma. The approach leverages a stable isotope internal standard and optimized SPE cleanup to achieve low volume sampling and high reproducibility on a SCIEX QTRAP® 6500+ with ExionLC™ AD system.

Methodology and Instrumentation

Sample Preparation:

• Aliquot 500 µL human plasma and spike with 50 µL barnidipine-D5 internal standard.
• Add 500 µL 2 % ortho-phosphoric acid and vortex.
• Perform SPE on Bond Elut Plexa cartridges: condition with methanol and water, load sample, wash with 1 % acetic acid in 5 % methanol then water, elute with acetonitrile.
• Evaporate under N2 and reconstitute in 200 µL mobile phase.

Chromatography:

• Column: Phenomenex Luna HILIC (150 × 4.6 mm, 3 µm)
• Mobile phase: 80 % 2 mM ammonium formate (pH 4), isocratic at 500 µL/min
• Column temperature: 40 °C, injection volume: 10 µL

Mass Spectrometry:

• Ionization: Positive ESI, IonDrive™ Turbo V source at 600 °C, curtain gas 30, GS1 60, GS2 60, spray voltage 3200 V
• Acquisition: MRM at unit resolution, dwell 200 ms, transitions m/z 492.2 → 315.1 for barnidipine and 497.2 → 315.1 for barnidipine-D5
• Data processing: Analyst® 1.6.3, 1/x2 weighted linear regression

Main Results and Discussion

The method exhibits:

• Linearity from 5 to 8 000 pg/mL (r² > 0.99 with 1/x2 weighting)
• Lower limit of quantification (LLOQ) at 5 pg/mL in plasma and LOD 0.1 pg/mL in a neat solution
• Recovery > 80 % across low, medium and high QC levels
• Precision within ±20 % CV at LLOQ and ±15 % CV at other QC levels

This performance supports reliable pharmacokinetic assessments with minimal sample volume and low reconstitution volume.

Benefits and Practical Applications

The assay requires only 500 µL plasma and provides high sensitivity, making it suitable for GLP-regulated studies, repeat injections and limited sample availability. It enables accurate PK profiling in early-phase clinical trials and therapeutic drug monitoring.

Future Trends and Opportunities

Advancements may include microflow LC-MS integration to reduce solvent consumption, automation of SPE workflows for higher throughput, and extension of the platform to multi-analyte panels. Coupling high-resolution MS could further enhance selectivity for complex biological matrices.

Conclusion

A robust and selective LC-MS/MS method for barnidipine quantification in human plasma was established on the SCIEX QTRAP 6500+ system. It meets stringent bioanalytical validation criteria while minimizing sample requirements, facilitating pharmacokinetic and clinical research.

References

1. Talari GP, Kumar VK. Simultaneous Estimation and Validation of LC-MS Method for Determination of Barnidipine in Human Plasma. RASAYAN J. Chem. 2019;12(1):389–401.
2. Delhira N, Anbazhagan S. A Simple, Isocratic and Ultra-Fast Liquid Chromatography/Mass Spectrometry Method for the Estimation of Barnidipine in Human Plasma. Pharm Anal Acta. 2015;6:7.