Fragmentation pathways of eight nitrogen-containing bisphosphonates (BPs) investigated by ESI-MS n in negative ion mode

Abstract

Fragmentation pathways of eight nitrogen-containing bisphosphonates (BPs), including Pamidronate, Alendronate, and five corresponding acylated derivatives, and Risedronate, were investigated by electrospray ionization mass spectrometry (ESI-MS) in conjunction with tandem mass spectrometry in negative ion mode. Characteristic fragment ions involved were formed by successive loss of water molecules, including α, β-unsaturated product ions formed by dehydration of the α-hydroxyl group and β-hydrogen, cyclic ions with P–O–P four-membered rings formed by dehydration of the two –OH groups attached to two phosphorus atoms, and cyclic phosphoramide ions with five- or six-membered rings formed by loss of water via an intramolecular nucleophilic substitution reaction, in which –NH 2 (or –NH–) group on γ or δ carbon acted as the nucleophile to attack phosphorus atom of P O group at the initial stage. Another notable characteristic product ion [HP 2O 5] − at m/ z 143, shown by all eight ESI-MS 2 spectra, is a diagnostic ion of bisphosphonate group, formed from the four-membered ring ion containing P–O–P. Some additional fragmentation ions were produced from chloroacetyl Alendronate, chloroacetyl Pamidronate, and Risedronate containing triple bond ions, formed by loss of water from the corresponding enol anions. The hydrogen/deuterium (H/D) exchange experiment, theoretical calculations, and the high-resolution mass spectrometry were appropriately employed to rationalize the proposed fragmentation pathways.