title: A robust method to quantify low molecular weight contaminants in heparin: detection of tris(2-n-butoxyethyl) phosphate.
abstract: Recently, oversulfated chondroitin sulfate (OSCS) was identified in contaminated heparin preparations, which were linked to several adverse clinical events and deaths. Orthogonal analytical techniques, namely nuclear magnetic resonance (NMR) and capillary electrophoresis (CE), have since been applied by several authors for the evaluation of heparin purity and safety. NMR identification and quantification of residual solvents and non-volatile low molecular contaminants with USP acceptance levels of toxicity was achieved 40-fold faster than the traditional GC-headspace technique, which takes ~120 min against ~3 min to obtain a (1)H NMR spectrum with a signal/noise ratio of at least 1000/1. The procedure allowed detection of Class 1 residual solvents at 2 ppm and quantification was possible above 10 ppm. 2D NMR techniques (edited-HSQC (1)H/(13)C) permitted visualization of otherwise masked EDTA signals at 3.68/59.7 ppm and 3.34/53.5 ppm, which may be overlapping mononuclear heparin signals, or those of ethanol and methanol. Detailed NMR and ESI-MS/MS studies revealed a hitherto unknown contaminant, tris(2-n-butoxyethyl) phosphate (TBEP), which has potential health risks.
authors.affiliation: Departamento de Bioquímica e Biologia Molecular, Universidade Federal do Paraná, Curitiba, PR CEP: 81531-980, Brazil.
authors.forename: Guilherme L
authors.initials: GL
authors.lastname: Sassaki
authors.forename: Daniel S
authors.initials: DS
authors.lastname: Riter
authors.forename: Arquimedes P
authors.initials: AP
authors.lastname: Santana Filho
authors.forename: Marco
authors.initials: M
authors.lastname: Guerrini
authors.forename: Marcelo A
authors.initials: MA
authors.lastname: Lima
authors.forename: Cesare
authors.initials: C
authors.lastname: Cosentino
authors.forename: Lauro M
authors.initials: LM
authors.lastname: Souza
authors.forename: Thales R
authors.initials: TR
authors.lastname: Cipriani
authors.forename: Timothy R
authors.initials: TR
authors.lastname: Rudd
authors.forename: Helena B
authors.initials: HB
authors.lastname: Nader
authors.forename: Edwin A
authors.initials: EA
authors.lastname: Yates
authors.forename: Philip A J
authors.initials: PA
authors.lastname: Gorin
authors.forename: Giangiacomo
authors.initials: G
authors.lastname: Torri
authors.forename: Marcello
authors.initials: M
authors.lastname: Iacomini
mesh_terms: D019075:Electrophoresis, Capillary / Q000379:methods; D006493:Heparin / Q000737:chemistry*; D009682:Magnetic Resonance Spectroscopy / Q000379:methods*; D010755:Organophosphates / Q000032:analysis*; D012997:Solvents / Q000737:chemistry
chemical_list: D010755:Organophosphates; D012997:Solvents; C012085:tris(2,3-dibromopropyl)phosphate; D006493:Heparin