12
May/June 2011
Hydrophilic Interaction Liquid Chromatography – A Potential Alternative for the Analysis of Dextran-1
Marc T. Barber* and Edward W. Hammersley Research Analytics, Pfizer Global Research and Development, Ramsgate Road, Sandwich, CT13 9NJ *Corresponding author. Email:
marc.barber@
pfizer.com
A method for the analysis of dextran-1 has been developed using hydrophilic interaction liquid chromatography (HILIC) with charged corona aerosol detection (CCAD) as a potential alternative to the size exclusion chromatography method described in the European Pharmacopoeia (EP). The EP method is in excess of 500 minutes and is considered long for routine analysis of multiple samples. The developed method retains resolution between monomers, whilst performing separation within 13 minutes.
Introduction Dextran is a complex mixture of
polysaccharides, principally of the α-1,6- glucan type, which is produced by the fermentation of sucrose by certain lactic acid bacteria, e.g. Leuconostoc mesenteroides and Streptococcus mutans. Clinical grade dextran is produced using Leuconostoc mesenteroidesstrain NRRL B-512 = CIP 78.59 or substrains thereof.1
The polymeric structure of dextran is shown in Figure 1.
Being a product of a fermentation process, dextran consists of different polymer lengths. Pharmacopoeial grade dextran is split into a number of different fractions, as defined in Table 1. The larger dextran fractions have been use clinically to reduce thrombosis.2
In
some patients the larger dextran fraction have the potential to induce anaphylactic reactions; this risk is reduced significantly when dextran-1 is pre-administered.3
The pharmacopoeial test for the average relative molecular mass is used as a quality property to define and control the overall distribution of glucose units present in the polymer length. The pharmacopoeial method uses size-exclusion chromatography (SEC)4 and has a run time in excess of 500 minutes. Based on the known capabilities of HILIC to retain highly polar analytes5
, a potential
alternative method, of 13 minutes, is described.
Figure 1: Structure of dextran
Component
Dextran-1 Dextran 40 Dextran 60 Dextran 70
Table 1: Dextran fractions Experimental
Samples and Reagents Purified water was produced from a Millipore Milli-Q system and was of 18.2 MΩcm and < 3ppb TOC. Acetonitrile (Chromasolv grade) was obtained from Sigma Aldrich, as was sodium chloride (ACS grade). Ammonium acetate
(Analytical puriss HPLC grade) was purchased from Fluka. Pharmacopoeial grade Dextran-1 was purchased from Pharmacosmos A/S.
Solutions were prepared by dissolving the appropriate amount of Dextran-1 in the appropriate method diluent (see Tables 2 and 3).
Average relative molecular mass ~1000
~40 000 ~ 60 000 ~70 000
Average relative molecular mass Range
850 to 1150
35 000 to 45 000 54 000 to 66 000 64 000 to 76 000
Page 1 |
Page 2 |
Page 3 |
Page 4 |
Page 5 |
Page 6 |
Page 7 |
Page 8 |
Page 9 |
Page 10 |
Page 11 |
Page 12 |
Page 13 |
Page 14 |
Page 15 |
Page 16 |
Page 17 |
Page 18 |
Page 19 |
Page 20 |
Page 21 |
Page 22 |
Page 23 |
Page 24 |
Page 25 |
Page 26 |
Page 27 |
Page 28 |
Page 29 |
Page 30 |
Page 31 |
Page 32 |
Page 33 |
Page 34 |
Page 35 |
Page 36 |
Page 37 |
Page 38 |
Page 39 |
Page 40 |
Page 41 |
Page 42 |
Page 43 |
Page 44 |
Page 45 |
Page 46 |
Page 47 |
Page 48 |
Page 49 |
Page 50 |
Page 51 |
Page 52
