(M1330-03-20) RP-HPLC Method Development for the Simultaneous Estimation of Trazodone and Gabapentin in Bulk Drug

Purpose: Up to 50% of diabetic patients with neuropathy experience chronic pain, known as painful diabetic neuropathy (PDN), a condition with a profound impact on quality of life and limited effective treatment options. Gabapentin is commonly prescribed for PDN but is often associated with dose-limiting side effects. Recent studies have indicated that trazodone, a multi-modal antidepressant, shows promise at low doses as an adjunct therapy to gabapentin. Based on clinical and preclinical evidence, a fixed-dose combination (FDC) of gabapentin and trazodone has been developed to address neuropathic pain more effectively while minimizing adverse effects. Notably, patients treated with gabapentin/ trazodone 2.5/25 mg TID demonstrated statistically significant pain relief [1]. The primary focus of this research is the development of a reverse-phase high-performance liquid chromatography (RP-HPLC) method for the simultaneous quantification of gabapentin/ trazodone. While several analytical methods are available in the literature for the individual estimation of gabapentin and trazodone, no RP-HPLC method currently exists for their simultaneous estimation. Therefore, this study aims to develop a novel, robust, and validated RP-HPLC method capable of accurately and reliably quantifying both drugs.

Methods: The chromatographic analysis was performed using an AGILENT 1100 HPLC system equipped with a diode array detector, prominence pump, column oven, LAB Solution software, and an automatic sample injector. A Phenomenex Luna C18 column (250 mm × 4.6 mm, 5 µm) was employed as the stationary phase for the separation of gabapentin and trazodone. The buffer solution was prepared by dissolving 2.72 g of potassium dihydrogen phosphate (KH₂PO₄) in a 1000 mL volumetric flask with Milli-Q water, adjusting the pH to 4 using orthophosphoric acid (OPA). The mobile phase was prepared by mixing 60 parts of this buffer with 40 parts of acetonitrile (ACN), and the final pH was adjusted to 6.8 using OPA. This mobile phase was sonicated for 15 minutes prior to use.The optimized experimental conditions included a mobile phase composition of phosphate buffer (pH 6.8) and acetonitrile in a 70:30 v/v ratio. The flow rate was maintained at 0.8 mL/min, with a column oven temperature of 30°C. Detection was carried out at a wavelength of 210 nm. A sample volume of 20 µL containing a gabapentin/trazodone 25/2.5 µg/mL mixture of trazodone and gabapentin was injected into the system. To ensure optimal performance and reproducibility of the chromatographic system, the column was pre-saturated for 90 minutes prior to analysis. A thorough column washing procedure was carried out for 240 minutes to maintain stability and prevent any carryover or contamination between runs. These conditions contributed to achieving reliable and consistent separation of the analytes.

Results: The developed RP-HPLC method successfully achieved the simultaneous estimation of gabapentin and trazodone with a total run time of less than 10 minutes. The retention time for trazodone was observed at 1.906 minutes, while gabapentin eluted at 2.503 minutes. The peak area recorded for gabapentin was 189.2 and for trazodone, 103.7. Symmetry factors were 1.35 for gabapentin and 1.004 for trazodone, indicating good peak shape for trazodone and slight fronting in the gabapentin peak. The resolution between gabapentin and trazodone was within acceptable limits, ensuring clear separation of both compounds. Although minor peak fronting was observed for gabapentin, the method demonstrated satisfactory performance across the tested concentration range (0.8 to 1.8 µg/mL), making it suitable for routine analytical and quality control purposes.

Conclusion: The proposed RP-HPLC method successfully achieved the resolution of gabapentin and trazodone within a runtime of less than 10 minutes. This method demonstrates suitability for routine laboratory analysis and can be effectively applied for the quality control of combined dosage formulations

References: 1) Tesfaye, S., Saravanan, P., Ehler, E. et al. Efficacy and Safety of Trazodone and Gabapentin Fixed-Dose Combination in Patients Affected by Painful Diabetic Neuropathy: Randomized, Controlled, Dose-Finding Study. Pain Ther 13, 987–1006 (2024). https://doi.org/10.1007/s40122-024-00624-3.

Acknowledgements: The authors express their gratitude to Arnold and Marie Schwartz College of Pharmacy and Health Sciences, Long Island University, Brooklyn, NY, USA—11201, USA, for providing the necessary resources and support to carry out the research successfully.