Simultaneous determination of β-methyl-β-nitrostyrolene and its related substances by PS-DVB chromatographic column and structural analysis of its photo-isomerization product

A method of high performance liquid chromatography (HPLC) was established to determine β-methyl-β-nitrostyrolene (MNS) and its related substances and to investigate the stability of MNS in acetonitrile solution. In this chromatographic method, MNS and its related substances were separated using the polystyrene-divinyl-benzene column (PS-DVB) (25 cm × 4.6 mm i.d. × 6 μm). Acetonitrile-water served as the mobile phase with gradient elution of 0-8 min, ACN 74-92%, maintaining the flow rate at 1.0 mL min^-1with the UV detection wavelength at 214 and 250 nm. The selected chromatographic conditions were found to effectively separate MNS, benzaldehyde and nitroethane with retention times of 3.66, 5.51 and 8.94 min, and perfect resolutions of 6.76 and 8.69. The linearity range of MNS, benzaldehyde and nitroethane were found in the range of 25-125, 0.16-15.71 and 0.16-15.67 μg mL^-1, with the limits of detection of 0.031, 0.006 and 0.011 μg mL^-1, respectively. Recovery studies showed good results for all examined compounds (from 96.5 to 99.9%) with RSD ranging from 0.31 to 0.94%. MNS was unstable in acetonitrile when exposed to natural light, and a new product appeared. The new product was purified and identified as α-methyl-styrolene nitrite by GC-MS and NMR, which was the photo-isomerization product of MNS. The proposed method was found to be accurate, precise, reproducible and specific and it could also be used for routine quality-control analysis of MNS.