Fast LC/MS/MS Method for Quantitative Determination of Omeprazole in Human Plasma

Importance of the Topic

Accurate and rapid measurement of drug concentrations in biological fluids is fundamental for pharmacokinetic studies, therapeutic monitoring and safety assessments. Omeprazole, a widely used proton pump inhibitor, requires sensitive quantification in plasma to support clinical trials and bioequivalence studies. This application note demonstrates a high-throughput LC-MS/MS protocol enabling precise measurement of omeprazole in human plasma, addressing the need for speed, selectivity and low detection limits in routine bioanalysis.

Objectives and Study Overview

The primary aim was to develop and validate a fast, robust UHPLC-MS/MS method for quantifying omeprazole levels in human plasma over a wide dynamic range. Key goals included:

• Minimizing sample preparation time through a simple liquid–liquid extraction.
• Establishing a lower limit of quantitation (LLOQ) of 1.0 ng/mL.
• Ensuring linearity from 1.0 to 2000 ng/mL with excellent accuracy and precision.

Applied Methodology and Instrumentation

Sample Preparation:

• Spike 180 μL of human plasma with standard or QC solutions.
• Add 200 μL of sodium bicarbonate buffer (pH 10.5), vortex for 60 seconds.
• Extract with 600 μL ethyl acetate, shake for 5 minutes followed by centrifugation.
• Collect 100 μL of organic layer, dilute with 200 μL acetonitrile and inject.

Chromatography and Mass Spectrometry:

• UHPLC column: Capcell pak C18 (50×2.0 mm, 3 μm) at 35 °C, flow rate 800 μL/min.
• Mobile phases: A) 0.02 M ammonium formate pH 3.0; B) acetonitrile:methanol (50:50).
• Gradient program: 5% B at 0.01 min, ramp to 95% B at 2.00 min, re-equilibrate by 3.50 min.
• MS detector: Triple quadrupole with ESI interface (4.5 kV), DL 220 °C, heat block 275 °C.
• MRM transitions: omeprazole m/z 346.00→198.10 (positive mode), tolbutamide (IS) m/z 269.00→170.10 (negative mode).

Main Results and Discussion

Linearity and Sensitivity:
Calibration curves showed linear response from 1.0 to 2000 ng/mL (weighted 1/x2 regression, R2 = 0.99987). The LLOQ of 1.0 ng/mL met criteria for precision (CV < 20%) and accuracy.

Precision and Accuracy:
QC samples at 3.0, 900 and 1800 ng/mL demonstrated intra-run precision (%CV) between 3.1% and 12.4% with accuracy ranging from 80.0% to 113.3% of nominal values.

Extraction Recovery:
Comparison between pre- and post-extraction spiked samples yielded recoveries of 86.0% to 114.4%, confirming efficient analyte isolation from plasma.

Benefits and Practical Applications

This method delivers several advantages for bioanalytical laboratories:

• High throughput with total run time under 4 minutes.
• Low sample volume requirement (180 μL plasma).
• Robust quantitation across a broad concentration range.
• Simplified sample preparation minimizing matrix interference.

Future Trends and Potential Applications

Advancements in high-resolution MS and microflow LC promise further improvements in sensitivity and selectivity. Integration of automated sample preparation and online extraction could streamline workflows. Similar approaches may be extended to other proton pump inhibitors or co-administered drugs for comprehensive pharmacokinetic profiling.

Conclusion

A rapid and sensitive UHPLC-MS/MS assay was successfully developed for omeprazole quantification in human plasma. The method meets rigorous bioanalytical validation criteria, offering reliable performance for pharmacokinetic and clinical studies.

Used Instrumentation

• UHPLC Nexera system
• Shimadzu LCMS-8040 Triple Quadrupole Mass Spectrometer

Reference

J. De Smet et al. Journal of Separation Science, 2010, 33, 939–947.