Determination of Pregabalin in Human Plasma by SPE-LC-MS/MS Using Thermo Scientific SOLA CX Mixed Mode Solid Phase Extraction Cartridges and a Thermo Scientific Accucore PFP HPLC Column

Importance of the Topic

A reliable and sensitive assay for pregabalin quantification in human plasma is essential for therapeutic drug monitoring in neuropathic pain, fibromyalgia and epilepsy management. Accurate measurement informs dosage adjustments, ensures patient safety and supports clinical pharmacokinetic studies.

Objectives and Study Overview

The aim of this work was to develop and validate a rapid solid-phase extraction coupled with liquid chromatography-tandem mass spectrometry (SPE-LC-MS/MS) method for determining pregabalin levels (1–250 ng/mL) using gabapentin as an internal standard. Emphasis was placed on high throughput, minimal solvent use and robust performance in a bioanalytical setting.

Methodology

• Sample Preparation: Human plasma (285 µL) was spiked with pregabalin and gabapentin, acidified and processed through SOLA CX SPE cartridges under positive pressure.
• Extraction Steps: Conditioning with methanol, equilibration with acidic water, sample loading, sequential acidic washes, and elution with basic methanol. Dried extracts were reconstituted in aqueous methanol with formic acid.
• Chromatography: Separation on a 50 × 2.1 mm Accucore PFP column (2.6 µm) at ambient temperature. Mobile phases were water and methanol, both containing 0.1% formic acid, run under a 5-minute gradient at 0.4 mL/min.
• Mass Spectrometry: Positive-mode heated electrospray ionization with selected reaction monitoring. Transitions monitored were m/z 160.1 → 55.1 for pregabalin and 172.1 → 95.1 for gabapentin.

Used Instrumentation

• Thermo Scientific Accela 1250 UHPLC pump and Open Autosampler
• Thermo Scientific TSQ Vantage triple quadrupole mass spectrometer
• Thermo Scientific SOLA CX SPE cartridges (10 mg, 1 mL)
• Thermo Scientific Accucore PFP HPLC column (50 × 2.1 mm, 2.6 µm)

Key Results and Discussion

The assay exhibited excellent chromatographic performance with retention times of 2.26 min for pregabalin and 2.17 min for the internal standard. Linearity over 1–250 ng/mL achieved an r2 of 0.999 using 1/x weighting. The lower limit of quantification of 1 ng/mL yielded signal-to-noise >198. Precision at a mid-range concentration (75 ng/mL) was 2.8% CV, recovery was 91.4% and matrix effect was –7.0%. Endogenous interferences eluted separately, confirming method specificity.

Benefits and Practical Applications

This SPE-LC-MS/MS approach offers rapid sample processing, low solvent consumption and robust quantitation suitable for high-throughput bioanalytical laboratories. The short run time and high sensitivity enable efficient therapeutic monitoring and pharmacokinetic profiling.

Future Trends and Opportunities

Advancements may include integration with automated SPE platforms, expansion to multiplexed assays for related anticonvulsants, and coupling with high-resolution mass spectrometry for comprehensive metabolite profiling. Miniaturized workflows and green chromatography initiatives will further enhance throughput and sustainability.

Conclusion

The validated SPE-LC-MS/MS method provides a reliable, fast and sensitive assay for pregabalin in human plasma, meeting regulatory criteria for linearity, accuracy, precision and specificity. Its simplicity and performance make it well suited for routine clinical and pharmacokinetic applications.

References

• U.S. National Library of Medicine. Pregabalin drug information. MedlinePlus.
• Therapeutic Advances in Chronic Disease. 2010;1(4):141–148.
• U.S. Food and Drug Administration. Guidance for Industry: Bioanalytical Method Validation.