Introduction: Gabapentin is an anticonvulsant drug prescribed for the treatment of neuropathic pain and seizures as well as for many off-label uses. Gabapentin is prescribed in high doses relative to other therapeutic drugs and is eliminated in urine predominantly in its unchanged form, which often results in extremely high concentrations of this compound occurring in patient urine samples. When analyzed by LC-MS/MS, high concentrations of gabapentin can have significant analytical implications, particularly for the compound amphetamine. Interference between gabapentin and amphetamine has been well documented and can result in signal suppression, poor peak shape, and shifting retention times. Other analytical challenges include saturation of the mass spectrometry detector and column overload. In this work, we explored several strategies to mitigate the effects of high gabapentin concentrations in urine samples.
Objectives: The objective of this study was to capture the analytical challenges presented by high concentrations of gabapentin in urine samples when analyzed by LC-MS/MS and investigate different strategies to mitigate them.
Methods: Using a Shimadzu 8045 LC-MS/MS system in ESI+ mode, a method developed for the analysis of 60 drugs of abuse in urine was used to test two samples: one containing 0.1 μg/mL of both gabapentin and amphetamine, and one containing 250 μg/mL of gabapentin and 0.1 μg/mL of amphetamine. After data collection, the signal of amphetamine was compared in each sample to determine if interference from gabapentin was occurring. The method utilized a Raptor Biphenyl 50 x 2.1 mm, 2.7 μm column with a mobile phase A of water and mobile phase B of methanol, both acidified with 0.1% formic acid. The flow rate was 0.6 mL/min, the column temperature was 45°C, and the injection volume was 5 μL. Gradient elution was employed, with a total run time of nine minutes. Following analysis, different strategies were tested to see if the interference between gabapentin and amphetamine could be resolved. These strategies included using alternate column lengths and diameters, decreasing injection volume, deoptimizing the analyte transition, and testing different mobile phase additives.
Results: Under the original method conditions tested, amphetamine showed a diminished signal and shifted retention time when a high concentration of gabapentin was present. The method was redeveloped using the strategies described. It was determined that the best approach for reducing interference was to fully chromatographically resolve gabapentin and amphetamine and ensure that gabapentin was the first compound to elute. This was done by switching the additive in mobile phase A from 0.1% formic acid to 10 mM ammonium formate, which affected the elution order of early eluting compounds. Resolution was further improved by switching from a 50 x 2.1 mm column to a 100 x 2.1 mm column. Detector saturation and column overload were improved by deoptimizing the mass transition for gabapentin and reducing the injection volume from 5 μL to 2 μL. Significant carryover was observed due to the high analyte concentrations and was eliminated by adding a small amount of 2-propanol to mobile phase B. Performance of the other analytes in the method was evaluated to ensure that they were not negatively affected by the change in method parameters.
Discussion: Interference between gabapentin and amphetamine, chromatographic overload, and detector saturation were all observed when analyzed under the original method conditions. Altering the mobile phase composition, using an extended column length, deoptimizing the mass transition, and reducing the injection volume were all successful in mitigating these analytical challenges. The addition of 2-propanol to mobile phase B helped to reduce carryover by washing contaminants off the analytical column more efficiently. The redeveloped method can be used to effectively analyze 60 compounds in urine without interference between gabapentin and amphetamine.
