title: Practical and economical implementation of online H/D exchange in LC-MS.
abstract: Structural elucidation is an integral part of drug discovery and development. In recent years, due to acceleration of the drug discovery and development process, there is a significant need for highly efficient methodologies for structural elucidation. In this work, we devised and standardized a simple and economical online hydrogen-deuterium exchange methodology, which can be used for structure elucidation purposes. Deuterium oxide (D2O) was infused as a postcolumn addition using the syringe pump at the time of elution of the analyte. The obtained hydrogen/deuterium (H/D) exchange spectrum of the unknown analyte was compared with the nonexchanged spectrum, and the extent of deuterium incorporation was delineated by using an algorithm to deconvolute partial H/D exchange, which confirmed the number of labile hydrogen(s) in the analyte. The procedure was standardized by optimizing flow rates of LC output, D2O infusion, sheath gas, and auxiliary gas using the model compound sulfasalazine. The robustness of the methodology was demonstrated by performing sensitivity analysis of various parameters such as concentrations of analyte, effect of matrices, concentrations of aqueous mobile phase, and types of LC modifiers. The optimized technique was also applied to chemically diverse analytes and tested on various mass spectrometers. Moreover, utility of the technique was demonstrated in the areas of impurity profiling and metabolite identification, taking pravastatin-lactone and N-oxide desloratidine, as examples.
authors.affiliation: Analytical Research and Development, Pharmaceutical Development, Biocon Bristol-Myers Squibb R&D Center, Syngene International Ltd. , Bangalore, India.
authors.forename: Ravi P
authors.initials: RP
authors.lastname: Shah
authors.forename: Amit
authors.initials: A
authors.lastname: Garg
authors.forename: Siva Prasad
authors.initials: SP
authors.lastname: Putlur
authors.forename: Santosh
authors.initials: S
authors.lastname: Wagh
authors.forename: Vineet
authors.initials: V
authors.lastname: Kumar
authors.forename: Venugopala
authors.initials: V
authors.lastname: Rao
authors.forename: Saranjit
authors.initials: S
authors.lastname: Singh
authors.forename: Sandhya
authors.initials: S
authors.lastname: Mandlekar
authors.forename: Sridhar
authors.initials: S
authors.lastname: Desikan
mesh_terms: D000818:Animals; D002853:Chromatography, Liquid / Q000379:methods*; D003903:Deuterium / Q000737:chemistry*; D006801:Humans; D006859:Hydrogen / Q000737:chemistry*; D007783:Lactones / Q000737:chemistry; D017336:Loratadine / Q000031:analogs & derivatives / Q000737:chemistry; D055432:Metabolomics; D008862:Microsomes, Liver / Q000378:metabolism; D009862:Online Systems; D010949:Plasma / Q000378:metabolism; D017035:Pravastatin / Q000737:chemistry / Q000302:isolation & purification; D051381:Rats; D021241:Spectrometry, Mass, Electrospray Ionization / Q000379:methods*; D016482:Urinalysis
chemical_list: D007783:Lactones; D017336:Loratadine; D006859:Hydrogen; D003903:Deuterium; C121345:desloratadine; D017035:Pravastatin