19
are not compatible with this mobile phase selection due to potential de-wetting/phase collapse [13]. Therefore, a polar embedded column that was compatible with 100% aqueous conditions was selected and assessed. A separation suitable to progress the projects development work was identified, (Figure 3), although upon extended use it became evident that the degree of resolution was not sufficient to produce a robust long term method and should impurities be present at elevated levels the separation obtained would no longer be baseline resolved which could lead to less accurate quantification.
Figure 3: The separation of 4 extremely polar compounds using AstraZeneca’s conventional method on a polar embedded C18 column [1].
Detector 1-195nm Final Robustness08
900 800
At this stage a PGC column was investigated to assess whether this column could yield a suitably robust separation of the polar analytes under reverse phase conditions. The mobile phase composition was also investigated on the PGC column, and a higher buffer concentration was found to be required to ensure good peak shape and good resolution between peaks 3 and 4.
700 600 500
Method 1: Test mix preparation
400 300 200 100 0 -100 0.0 0.2 0.4 0.6 0.8 1.0 1.2 1.4 1.6 1.8 2.0 2.2 2.4 2.6 Minutes 2.8 3.0 3.2 3.4 3.6 3.8 4.0 4.2 4.4 4.6 4.8 5.0
Approximately 3.00mg of compounds 1, 2, and 3 were dissolved in 100mL of sample diluents [water / acetonitrile (95:5, v/v)] to give a concentration of 0.03mg/mL impurity solution. Then 15.0mg of compound 4 was dissolved with 1mL impurity solution to give the final ratio of 0.2% impurity to parent compound (4).
Figure 4: The separation of 4 extremely polar compounds on Hypercarb 5 µm, 100 x 2.1mm column (method 1).
800
Detector 1-195nm Final Robustness05
Detector 1-195nm Final Robustness20
Detector 1-195nm Final Robustness25
Detector 1-195nm Final Robustness40
800
600
600
400
400
200
200
0 0.00 0.25 0.50 0.75 1.00 1.25 1.50 1.75 2.00 2.25 2.50 Minutes Figure 5. Overlaid chromatograms showing consistency and method 1 robustness on Hypercarb 5 µm, 100 x 2.1mm column. 2.75 3.00 3.25 3.50 3.75 4.00 4.25 4.50 4.75
0
5.00
mAU
mAU
mAU
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