Characterization of Currently Marketed Heparin Products: Reversed-Phase Ion-Pairing Liquid Chromatography Mass Spectrometry of Heparin Digests

Abstract

Here we report results from the analyses by enzymatic digestion and reversed-phase ion-pairing liquid chromatography mass spectrometry (RPIP-LC-MS) of active pharmaceutical ingredient (API) unfractionated heparins (UFHs) from six different manufacturers and one USP standard sample. We employed a reverse phase ion-pairing chromatography method using a C(18) column and hexylamine as the ion-pairing reagent with acetonitrile gradient elution to separate disaccharides generated from the digestion of the heparins by lyase I and III (E.C. 4.2.2.7 and 4.2.2.8) before introduction into an ion-trap mass spectrometer by an electrospray ionization (ESI) interface. Extracted ion chromatograms (EICs) were used to determine the relative abundance of the disaccharides by mass spectrometry. Eight disaccharides were observed and a similar composition profile was observed from digests of 20 UFH samples. The compositional profile determined from these experiments provides a measure of the norm and range of variation in "good" heparin to which future preparations can be compared. Furthermore, the profile obtained in the RPIP-LC-MS assay is sensitive to the presence of the contaminant, oversulfated chondroitin sulfate A (OSCS), in heparin.

Characterization of currently marketed heparin products: Analysis of heparin digests by RPIP-UHPLC–QTOF-MS

“…All the disaccharides, including II-H/III-H and II-S/III-S which were not resolved by RPIP-HPLC in our previous work [26], were either separated by UHPLC chromatography or by exact mass in extracted ion chromatograms. The split peaks observed for IV-A and III-S are attributed to resolved ␣-and ␤-anomers [33] (Fig.…”

“…For example, our laboratory reported conditions where the combined use of relatively high amounts heparin lyase I and III were effective for complete digestion of heparin sodium [26]. By contrast, Xiao et al observed that heparin lyase II yielded more efficient digestion than treatment with heparin lyase I or III alone and that applying lyase III alone did not provide efficient heparin digestion [9].…”

“…This work Sigma, Iduron II, III 5 mIU each for 60 g, 37 • C overnight Brustkern et al [26] Sigma I, III 10 mIU each for 40 g, 37 • C overnight Korir et al [17] Sigma I, II, III 10 mIU each for 20 g, 37 • C 17 h Zhang et al [18] Home-made I, II, III 10 mIU each for 5 g, 37 • C 10 h Galeotti and Volpi [19] Seikagaku I, II, III 1 IU each for 100 g, 37 • C 8 h Saad and Leary [20] Calbiochem I, II, III 10 mIU each for 20 g, 37 • C overnight Saad et al [21] Sigma, Calbiochem I, II, III 10 mIU each for 100 g, 37 • C 16 h Kinoshita and Sugahara [22] Seikagaku I, II, III 1.7 mIU-I and 0.85 mIU-III a for 1 g, 37 • C 30 min Camara et al [23] Sigma I, II, III 10 mIU each for 10 g, 37 • C 24 h Ruiz-Calero et al [24] Sigma I, II, III 13 mIU-I, 1.7 mIU-II, 1.5 mIU-III for 100 g, 37 • C 24 h Militsopoulou et al [25] Sigma, Seikagaku I, II, III 300 mIU each for 1 mg, 37 • C 90 min a Heparin lyase III contains a small proportion of lyase II. of 20 mM ammonium acetate and 2 mM calcium acetate (pH 7.4) and subjected to enzymatic digestion by adding different combinations of heparin lyase I, II or III dissolved in same buffer.…”

“…As previously described [26], heparin sodium or LMWH APIs were desalted via pressure dialysis with a 1000 Da molecular weight cutoff filter and then lyophilized. Sixty g of desalted heparin (10 L from 6 mg/mL water solution) was diluted with 40 L Table 1 Summary of enzymatic heparin digest protocols used.…”

“…Our previous work [26] found that hexylamine (HA) was optimal for separating heparin disacchrides with conventional ion-pairing reverse-phase HPLC before introduction into an ion-trap mass spectrometer. Heparin disaccharides in a mixture of eleven individual standards or the products of a complete digestion of heparin were injected onto an UHPLC system for separation.…”