Chiral detection of entecavir stereoisomeric impurities through coordination with R-besivance and ZnII using mass spectrometry

In this study, a mass spectrometry (MS)-based kinetic method (KM) is shown to be successful at analyzing a multichiral center drug stereoisomer, entecavir (ETV), both qualitatively and quantitatively. On the basis of the KM, the bivalent complex ion [M-II(A)(ref*)(2)](2+) (M-II=divalent metal ion, A=analyte, and ref*=chiral reference) was set as precursor ion in MS/MS. The experiment results suggest strong chiral selectivity between ETV and its isomers when using Zn-II coordinated with the chiral reference R-besivance (R-B). The logarithm of the fragment ion abundance ratio and the enantiomeric percentage (%) exhibits a strong linear relation because of the competitive loss of the reference and analyte. The product ion pair [Zn-II(R-B)A-H](+) (m/z 733) and [Zn-II(R-B)(2)-H](+) (m/z 849), together with [R-B+H](+) (m/z 394) and [A+H](+) (m/z 278), can realize the identification of ETV and all of its chiral isomers. Theoretical calculation were also performed using the B3LYP functional with the 6-31G* and LanL2DZ basis set to clarify the mechanism of structural difference of these bivalent complex ions. The results reveal that MS-KM can be used to detect optical impurities without a chiral chromatographic column and fussy sample pretreatment. The established method has been used to determine stereoisomeric impurities of less than 0.1% in ETV crude drug, a demonstration of its simple and effective nature for rapid detection of stereoisomeric impurities.