13


Figure 2: Typical SEC chromatogram for Dextran-1 150 mg/mL Solution for Injection (full and expanded scale)


Column: Flow:


Temperature:


Injection Volume: Detection:


Mobile Phase Sample Concentration


2 × Superdex Peptide 10/300 GL, 10 mm × 30 cm column 0.08 mL/min


20ºC to 25ºC 100 µL


Refractive index at a temperature of 30ºC 2.92 g/L sodium chloride


~ 6mg/mL Dextran-1 in 2.92 g/L sodium chloride (aq)


Table 2: Chromatographic conditions for pharmacopoeial method for Dextran-1 by Size-exclusion Chromatography Column:


Flow: Temperature:


Injection Volume: Detection:


Mobile Phase A: Mobile Phase B: Mobile Phase C: Gradient:


25ºC 20 µL


CAD with 100 pA range Water


Acetonitrile


100 mM Ammonium acetate (aq) Time (Minutes) % A % B % C


0


30 65 5


Sample Concentration Table 3: Chromatographic conditions for HILIC method for Dextran-1 Instruments and Methods


European Pharmacopeia Methodology The size exclusion method following the European Pharmacopeia method for determination of average molecular weight by SEC was run on an Agilent 1100 system equipped with a Waters 2414 Refractive Index Detector, using the conditions presented in Table 2.


HILIC Methodology


The HILIC method was developed using basic starting method development conditions for the Phenomenex Luna HILIC columns6


; and


run on an Agilent 1100 system equipped with ESA Corona Plus Charged Aerosol Detection. Modifications to the gradient were used to improve the separation between the glucose


units. The final chromatographic conditions are presented in Table 3.


Results and Discussion Typical chromatograms obtained for dextran-1 using the pharmacopoeial size exclusion methodology and the alternative HILIC methology are present in Figure 2 and Figure 3 respectively.


With the pharmacopoeial size exclusion method for dextran-1, the last peak to elute (glucose) was found to have a retention time of approximately 459 minutes, with each successive polymer unit eluting prior to the previous. This is as expected with SEC, as the smaller the molecule, more retention is experienced as it can penetrate more


− Average molecular mass (Mw): between 850 and 1150


− Fraction with less than 3 glucose units: less than 15%


− Fraction with more than 9 glucose units: less than 20%


Conclusion


The 13 minute HILIC method demonstrates a similar molecular mass distribution pattern for dextran-1. There is potential that this method may be a suitable alternative, to the pharmacopoeial method for the determination of the molecular mass distribution of dextran 1, though further work may be required to understand the differences between the two methods.


7


40 55 5


7.1 30 65 5


~ 4.5 mg/mL Dextran-1 in water/acetonitrile 35/65 v/v


13 30 65 5


Phenomenex Luna HILIC 3µm 150 mm × 4.6 mm column 1.0 mL/min


through the solid phase pores, whilst the larger the molecule becomes the less interaction it has with the solid phase pores and will elute earlier.


Using the HILIC column, the first peak to elute is glucose, and whilst full baseline separation is not achieved, the chromatography is acceptable. With a pore size of 200 Å, there is potential for some size exclusion effect, however, the change in elution order suggests that the hydrophilic interaction is the dominant effect.


As a short comparison, the molecular weight distribution for the dextran-1 was determined for both methodologies using the pharmacopoeial equation (Equation 1). The results for the molecular weight fractions and the average molecular mass are presented in Table 4.


Equation 1 MW


= average molecular mass of Dextran-1 wi= molecular mass of oligosaccharide i mi= weight proportion of oligosaccharide i


The data shows minor differences in the molecular weight distribution for the dextran-1. By both methods the sample meets the pharmacopoeial criteria in the monograph of:


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