Background: Pregabalin has been used for the treatment of pain. A clinically accepted method applied to patients with pain has not been published for the determination of pregabalin in human plasma. This study developed a fluorometric ultrahigh-performance liquid chromatography (UHPLC) method to measure pregabalin concentration in patients with pain.
Methods: After plasma pretreatment involving protein precipitation, pregabalin and gabapentin as an internal standard were derivatized with 4-fluoro-7-nitrobenzofurazan (NBD-F) under the following reaction conditions: 1 minute, pH 10, and 60°C. The UHPLC separation was performed using a 2.3-μm particle size octadecylsilyl column. The fluorescence detector was set at excitation and emission wavelengths of 470 and 530 nm, respectively. The predose blood samples were collected from 40 patients with pain who have been treated with 75 mg of pregabalin twice daily.
Results: The chromatographic run time was 1.25 minutes. No interfering peaks were observed in the blank plasma at the retention times of NBD derivatives. The calibration curve of pregabalin was linear at a range of 0.05-10 mcg/mL (r > 0.999). The lower limit of quantification was 0.05 mcg/mL. The intra-assay accuracy and precision were 98.3%-99.8% and within 4.3%, respectively. The inter-assay accuracy and precision were 103.2%-107.1% and within 4.1%, respectively. The predose plasma concentration of pregabalin in patients with pain ranged from 0.14 to 8.5 mcg/mL.
Conclusions: This study provides a validated fluorometric UHPLC method with fast analytical performance for the determination of pregabalin in human plasma. The present method could be applied to patients with pain and be used for the clinical research or therapeutic drug monitoring of pregabalin.