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.

Glycan analysis of Fonsecaea monophora from clinical and environmental origins reveals different structural profile and human antigenic response

“…Sucrose consumption was determined by Silica-Gel 60G TLC plates (Merck), with sucrose solutions in different concentrations as standard (2.5–10 mg.ml −1 ). Plates were developed with AcOEt: n -PrOH:AcOH:H 2 O (4:2:2:1, v/v), and detection of carbohydrates was performed with orcinol-H 2 SO 4 treatment (Sassaki et al, 2011). The plates were analyzed by densitometry using the Scion Image Program (Scion Corporation, Maryland, USA) and the results plotted in Microsoft Excel 2007 (Sassaki et al, 2005).…”

“…Integration of H-1 area was performed without tube rotation and respecting a HDO signal with a medium half line width varying from 2.5 to 3.5 Hz after Lorentzian deconvolution and post Fourier transformation. 2D NMR experiment was carried out using HSQC, heteronuclear correlation via double inept transfer with decoupling during acquisition, using trim pulses in inept transfer (hsqcetgpsi) program recorded for quadrature detection in the indirect dimension and acquired using 32 scans per series of 2 K × 400 data points, with zero filling in F1 (4 K) prior to Fourier transformation (Sassaki et al, 2011). …”

Update of the recommendations on biosimilar low‐molecular‐weight heparins from the Scientific Subcommittee on Control of Anticoagulation of the International Society on Thrombosis and Haemostasis

“…However, this number of oligosaccharides is lower than what is statistically predictable [19]. Furthermore, impurities in a biosimilar LMWH may remain from the start of the process or may be added to the preparations [19][20][21][22].…”

“…To ensure the correct sequences of the oligosaccharides of a biosimilar vs. the originator LMWH, the oligosaccharides have to be recomposed using accepted mapping strategies from the bulk of originator and biosimilar disaccharides. 6 Other glycosaminoglycans or impurities, detected by nuclear magnetic resonance and other techniques, should not be allowed [19][20][21][22]. No significant differences between biosimilar and originator LMWHs should be found by repeated analysis of one lot of the LMWHs as well as for different lots of both LMWHs.…”

GAG-ID: Heparan Sulfate (HS) and Heparin Glycosaminoglycan High-Throughput Identification Software*

“…Heparin and heparan sulfate (HS) 1 are involved in numerous physiological (1) and pathophysiological (2) processes, including cellular and organ development (3,4), cancer (5,6), and angiogenesis (7). Furthermore, heparin and heparan sulfate have been linked to regulation of cell growth (8), cell adhesion (9), inflammation and immune cell migration (10), neural development and regeneration (11,12), and hemostasis (13).…”