LC-MS/MS Method for the Determination of Tenofovir from Plasma

Importance of Topic

Quantitative analysis of tenofovir, a widely used nucleotide analogue reverse transcriptase inhibitor for HIV treatment, is essential for pharmacokinetic studies, therapeutic monitoring, and clinical research. Reliable, high-throughput methods support drug development, dosing optimization, and quality assurance in bioanalytical laboratories.

Objectives and Overview

This study describes the development and validation of a rapid, sensitive, and reproducible LC-MS/MS method for determining tenofovir concentrations in human plasma. Key aims include minimizing sample preparation time, achieving low limits of quantification, and ensuring robust chromatographic performance across a wide dynamic range.

Methodology and Instrumentation

Sample Preparation

• SPE sorbent: Thermo Scientific SOLA CX 96-well plate (10 mg/2 mL)
• Conditioning: 0.5 mL methanol followed by 0.5 mL water
• Loading: 200 µL plasma spiked with tenofovir and acidified with 1% formic acid
• Wash steps: 500 µL 1% formic acid in water, then 500 µL 1% formic acid in methanol
• Elution: 2×250 µL 20% ammonia in methanol, evaporation, and reconstitution in water/methanol (90:10, v/v)

Liquid Chromatography

• System: Thermo Scientific Accela 600 UHPLC
• Column: Hypersil GOLD, 1.9 µm, 50×2.1 mm
• Mobile phases: 25 mM ammonium acetate (pH 5) and methanol
• Gradient: 0→100% B in 1 min, flow rate 0.8 mL/min, column at 40 °C

Mass Spectrometry

• Instrument: Thermo Scientific TSQ Vantage MS
• Ionization: HESI, positive mode
• MRM transition: m/z 288.1→176.2, collision energy 24 eV
• Spray voltage 3000 V, vaporizer 350 °C, capillary 300 °C

Data Processing: Thermo Scientific LCQUAN software

Main Results and Discussion

The method achieved a limit of quantification of 1 ng/mL and linear response from 1 to 1000 ng/mL (r²=0.998). Representative chromatograms showed tenofovir eluting at ~0.8 min with excellent peak symmetry and baseline resolution from endogenous interferents. Recovery was 88.5%, and inter-day precision at 500 ng/mL was <3.7% CV, demonstrating high accuracy and reproducibility without requiring an internal standard.

Benefits and Practical Applications

• Rapid sample throughput with 3‐minute cycle time per injection
• Simplified SPE workflow minimizing solvent use
• High sensitivity for low-level quantification in clinical and pharmacokinetic studies
• Cost savings by eliminating the need for expensive internal standards and extensive cleanup

Future Trends and Opportunities

Advancements may include integration of automated SPE platforms for greater throughput, exploration of novel sorbent chemistries to further enhance selectivity, coupling with high-resolution MS for broader metabolite profiling, and miniaturized LC-MS systems for point-of-care therapeutic monitoring.

Conclusion

The described LC-MS/MS method using SOLA CX SPE and Hypersil GOLD UHPLC provides a fast, robust, and sensitive approach for tenofovir quantification in plasma, meeting the needs of high-throughput bioanalysis and clinical research.

References

• Phipps K. Thermo Fisher Scientific Application Note 20687, 2013.