ORIGINAL PAPER NMR of heparin API: investigation of unidentified signals in the USP-specified range of 2.12–3.00 ppm Sarah E. Lee & Edward K. Chess & Barrett Rabinow & G. Joseph Ray & Christina M. Szabo & Bennett Melnick & Reagan L. Miller & Lakshmy M. Nair & Edwin G. Moore Received: 20 July 2010 / Revised: 24 September 2010 / Accepted: 28 September 2010 / Published online: 15 October 2010 # Springer-Verlag 2010 Abstract This article addresses the identification and quantification of the chemical species resulting in reso- nances at 2.17 and 2.25 ppm in the 1 H nuclear magnetic resonance (NMR) spectrum of pharmaceutical-grade hepa- rin sodium. The NMR signals in question were first confirmed to arise from chemical moieties covalently attached to the heparin molecule through NMR diffusion experiments as well as chemical treatment of heparin active pharmaceutical ingredient (API) containing the resonances. The material responsible for the extra NMR signals was then demonstrated by NMR spiking studies to be something other than oversulfated chondroitin sulfate and was finally identified as an O-acetylation product of heparin through 13 C labeling experiments with subsequent NMR analysis. The extent of O-acetylation was quantified using three orthogonal techniques: 1 H NMR, ion chromatography, and headspace gas chromatography/mass spectrometry. The results of this work showed good agreement between the three quantitative methods developed to analyze the signals in the United States Pharmacopeia-specified region of 2.12–3.00 ppm for heparin API. Keywords Heparin . Acetylation . NMR . Isotopic labeling Introductory statement Heparin is a polydisperse mixture of glycosaminoglycans and has been the primary anticoagulant in clinical use for over 75 years for a number of life-saving medical therapies, including hemodialysis and cardiopulmonary bypass graft surgery, due to its efficacy, low cost, and the ability to rapidly reverse its anticoagulant activity [1–3]. The heparin molecule is a linear repeating disaccharide structure of 1,4- linked glucosamine and hexuronic acid residues (more specifically, derivatives of α-D-glucosamido (N-sulfated, O- sulfated, or N-acetylated) and O-sulfated uronic acid (α-L- iduronic acid or β-D-glucuronic acid)) [4–6]. Heparin is prepared by isolation from animal tissue, most commonly porcine intestine, by extraction followed by an extensive cleanup procedure that incorporates multiple precipitation and viral inactivation steps. In late 2007 and early 2008, a cluster of adverse events in patients receiving heparin sodium injections occurred in the USA and in some countries in Europe. The adverse events were reported as being “allergic-type” reactions, mainly characterized by acute hypotension, nausea, and shortness of breath. The root cause of these certain adverse events was determined to be a contamination of heparin by oversulfated chondroitin sulfate (OSCS) [7–11]. In order to ensure the availability and safety of this important drug, the FDA posted two methods of screening for the presence of OSCS in heparin [12] and required all US manufacturers of pharmaceuticals containing heparin to use these methods to screen their heparin sodium active pharmaceutical ingre- dients (APIs). The United States Pharmacopeia (USP) quickly incorporated these two methods, a capillary electrophoresis method and a 1 H nuclear magnetic reso- nance (NMR) method, into a proposed interim revision of the Heparin Sodium monograph [13], and the NMR method Published in the special issue Heparin Characterization with Guest Editor Cynthia K. Larive. S. E. Lee : E. K. Chess : B. Rabinow : G. J. Ray : C. M. Szabo : B. Melnick : R. L. Miller : L. M. Nair : E. G. Moore (*) Baxter Healthcare, WG3-3S, 25212W, Illinois Rte. 120, Round Lake, IL 60073, USA e-mail: ed_moore@baxter.com Anal Bioanal Chem (2011) 399:651–662 DOI 10.1007/s00216-010-4262-y