A High Sensitivity Method for the Quantifi cation of Fluvastatin in Plasma Using an Agilent 1290 Infi nity Binary LC and an Agilent 6490 Triple Quadrupole Mass Spectrometer

Significance of the Topic

The quantification of fluvastatin in human plasma is critical for pharmacokinetic studies and therapeutic drug monitoring. Its rapid metabolism and low plasma concentrations demand high sensitivity and specificity in bioanalytical assays.

Objectives and Overview of the Study

This application note reports the development of a robust LC–MS/MS method to quantify fluvastatin in 200 μL plasma samples. The study employed a deuterated analog of fluvastatin (fluvastatin-d6) as an internal standard to ensure accurate quantitation across a calibration range of 0.2 to 50 ng/mL and assessed method performance parameters including sensitivity, linearity, precision, accuracy, and carryover.

Methodology and Instrumentation

Sample preparation involved protein precipitation with ice-cold acetonitrile, followed by centrifugation, vacuum concentration, and reconstitution in an 80:20 mixture of aqueous ammonium acetate and acetonitrile. Chromatographic separation was achieved on a phenyl-hexyl column (2.1 × 50 mm, 1.8 μm) with a rapid gradient from 30% to 90% acetonitrile in 2 minutes. Detection was performed in positive multiple reaction monitoring mode.

Instrumentations Used

• Agilent 1290 Infinity Binary LC System (Flexible Cube, Binary Pump, Autosampler, Thermostat, TCC)
• Agilent 6490 Triple Quadrupole Mass Spectrometer

Main Results and Discussion

The method achieved a limit of detection of 0.1 ng/mL and a lower limit of quantitation of 0.2 ng/mL. Calibration curves demonstrated excellent linearity (r2 > 0.995) with 1/x weighting across eight levels. Intra- and interday accuracy and precision at LLOQ, low, medium, and high QC levels were within ±15% and %RSD below 15%. Negligible carryover was confirmed by overlaying chromatograms of the LOD sample and the first blank after the upper limit of quantitation.

Benefits and Practical Applications

With a 2-minute run time and straightforward protein precipitation, this assay supports high-throughput analysis in clinical research and pharmacokinetic profiling. Use of the deuterated internal standard minimizes matrix effects and enhances quantitation reliability.

Future Trends and Applications

Ongoing developments could integrate automated or microfluidic sample preparation to further boost throughput and reduce solvent use. Coupling with high-resolution mass spectrometry may enable simultaneous measurement of multiple statins and their metabolites for comprehensive lipid-lowering drug studies.

Conclusion

The presented LC–MS/MS method offers high sensitivity, precision, and robustness for routine quantification of fluvastatin in human plasma. Its rapid cycle time and reproducible performance make it well suited for pharmacokinetic and therapeutic monitoring applications.

References

1. U.S. Food and Drug Administration. Guidance for Industry: Bioanalytical Method Validation; 2001.
2. Kailasam S. Determination of fluvastatin in plasma using Agilent 6410B Triple Quadrupole LC/MS and 1200 Series Rapid Resolution LC. Agilent Technologies Application Note 5989-9751EN.