Reversed-phase ion-pair ultra-high-performance-liquid chromatography–mass spectrometry for fingerprinting low-molecular-weight heparins

Heparin is a complex mixture of sulfated linear carbohydrate polymers. It is widely used as an antithrombotic drug, though it has been shown to have a myriad of additional biological activities. Heparin is often partially depolymerized in order to decrease the average molecular weight, as it has been shown that low molecular weight heparins (LMWH) possess more desirable pharmacokinetic and pharmacodynamic properties than unfractionated heparin (UFH). Due to the prevalence of LMWHs in the market and the emerging availability of generic LMWH products, it is important that analytical methods be developed to ensure the drug quality. This work explores the use of tributylamine (TrBA), dibutylamine (DBA), and pentylamine (PTA) as ion-pairing reagents in conjunction with acetonitrile and methanol modified mobile phases for reversed-phase ion-pairing ultraperformance liquid chromatography coupled to mass spectrometry (RPIP-UPLC–MS) for fingerprint analysis of LMWH preparations. RPIP-UPLC–MS fingerprints are presented and compared for tinzaparinand enoxaparin.

► Dibutylamine and MeOH are effective for resolving ultra-low-molecular-weight heparin.
► Pentylamine and ACN is an effective combination for fingerprinting LMWHs.
► Fingerprints of Tinzaparin and Enoxaparin are compared for oligosaccharide composition.
► Clear differences are observed between LMWH preparations using RPIP-UPLC–MS fingerprinting.