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Previously, the FDA validated a method to assess the structure and composition of heparin products by separating and quantifying disaccharide level digests by reverse-phase-ion-pairing liquid chromatography (RPIP-HPLC) coupled to a low resolution and low sensitivity ion trap mass-spectrometer. Here, improved separation, information content and sensitivity were obtained through the use of reverse phase ion-pairing ultra-high pressure liquid chromatography (RPIP-UHPLC) coupled with a quadrupole time-of-flight (Q-TOF) mass spectrometer. Thus, with the new method, improved structural characterization of the same 20 lots of heparin sodium active pharmaceutical ingredients (APIs) as were analyzed in the previous work were obtained. In addition, for the first time, 10 low molecular weight heparin (LMWH) lots were characterized representing multiple lots manufactured by three different processes (dalteparin, tinzaparin or enoxaparin). In this study, UHPLC separation conditions and the enzymatic digesting protocol were optimized for analysis of disaccharide level digests of heparin and positive and negative electrospray ionization (ESI) modes were tested. The negative ion mode ESI analysis was found to be superior to the positive ion mode for these measurements, and a combination of heparin lyase II and III were optimal for heparin digestion. The data obtained establishes the normal variation in the composition of heparin sodium or LMWHs in this assay. These values are useful as possible product benchmarks and for surveillance of the heparin products being imported into the US market., (Published by Elsevier B.V.)