Development of an ion-pairing reversed-phase liquid chromatography method using a double detection analysis (UV and evaporative light scattering detection) to monitor the stability of Alimta ®-pemetrexed preparations: Identification and quantification of l-glutamic acid as a potential degradation product

Abstract

A new method based on high-performance liquid chromatography coupled to ultraviolet and evaporative light scattering detection (HPLC-UV-ELSD) was developed for the determination of l-glutamic acid, a potential degradation product of pemetrexed, and for the quantification of pemetrexed itself. This is an ion-pairing, reversed-phase method. The column was a Synergi MAX-RP C12 4 μm (150 mm × 4.6 mm). The mobile phase was 1 mM tridecafluoroheptanoic acid in aqueous solution and acetonitrile under gradient elution mode. l-Glutamic acid was detected by ELSD, and pemetrexed by UV at 254 nm. Good resolution was achieved between pemetrexed and l-glutamic acid. The HPLC method was validated according to SFSTP and ICH guidelines, and applied the accuracy profile procedure with a five-level validation experimental design. For pemetrexed, the decision criteria selected consisted of the acceptability limits (±3%) and the proportion of results within the calculated tolerance intervals (95%). In conclusion, the proposed analytical procedures were validated over the selected validation domains for l-glutamic acid (0.005–0.025 mg/mL) and pemetrexed (0.4–0.6 mg/mL) and shown to provide a very effective method.