Determination of eight isomers and related substance of Aprepitant using normal-phase and reverse-phase HPLC methods with mass spectrophotometric detection
Introduction: Aprepitant is a chiral prodrug possessing three stereogenic centers and exists in eight stereochemical forms. It was aimed to estimate and validate Aprepitant using reverse phase HPLC. Materials and methods: Chiral liquid chromatography with an ion-trap mass spectrophotometer was used to isolate all the isomers. The separation of the eight isomers was achieved on an amylase-based chiral column (Chiralpak ADH, 250 mm 4.6 mm) using n-hexane/isopropyl alcohol/methanol/trifluoroacetic acid (970/40/4/0.5, vol/vol/vol/vol) as the mobile phase at a flow rate of 0.5 ml/min. A reverse-phase HPLC method was used to analyze Aprepitant and its related substances. The determination of Aprepitant and its related substances was developed by using Xterra RP e 18, 250 mm 4.6 mm, 5 mm column. Results and conclusion: These methods were validated according to the International Conference on Harmonization (ICH) guidelines. The limit of detection (LOD) and limit of quantitation (LOQ) for Aprepitant were found to be 0.14 mg/ml and 0.41 mg/ml, respectively, for chiral substance and 0.07 mg/ml and 0.21 mg/ml, respectively, for the related substance. Key words: Aprepitant, Chiral separation, Related substances, HPLC Mass spectrophotometer.