Development and validation of a chiral LC-ESI-MS/MS method for simultaneous determination of the enantiomeric metabolites isosorbide 2-mononitrate and isosorbide 5-mononitrate of isosorbide dinitrate in rat and human plasma

Abstract A specific liquid chromatography– tandem mass spectrometry(LC-ESI-MS/MS) assay was developed and validated for simultaneous determination of two active metabolites of isosorbide dinitrate(ISDN), namely, isosorbide2-mononitrate (IS 2-MN) and isosorbide 5-mononitrate(IS 5-MN). A simple protein precipitationextraction technique was employed using 13 C 6 isosorbide 5-mononitrateas the internal standard. The two isomers were separated on a chiral column and mass detection was carried out by electrospray ionization(ESI) in negative multiple reaction monitoring (MRM) mode (ESI -ve). As neutral organic nitrates do not ionize well in ESI ion source, adduct formation of IS 2-MN and IS 5-MN were evaluated. Acetate adduct ions of IS 2-MN and IS 5-MN were well ionized and fragmentable in the negative mode by liquid chromatography electrospray ionization/ tandem mass spectrometry. These acetates adduct ions, of IS 2-MN and IS 5-MN were selected as parent mass for quantitation. The method was developed and validated in rat and human plasma with K 2 EDTA as an anticoagulant. This simultaneous quantitation method was shown to be linear over a working rangeof 25.0 ng/mL to 5050 ng/mL and 12.4 ng/mL to 2500 ng/mL for IS 2-MN (r 2  > 0.99) and IS 5-MN (r 2  > 0.99), respectively, in rat and human plasma. Sensitivity was determined as 25.0 ng/mL for IS 2-MN and 12.4 ng/mL for IS 5-MN in rat and human plasma. Inter- and intra-day accuracy and precision were within ±15% in both method validations. This validated method was subsequently applied to a pharmacokinetic (PK) study of ISDN in rat after oral administration.