18 August / September 2016

in these experiments is approximately 1.33 g/l. Thus, only a small part of the material was dissolved in the extraction cartridge. In the 10 second initial injection, roughly 13 mg of warfarin were injected from the saturated solution in the extractor. In contrast, the impurity level in the sample was low and the impurities would not be expected to reach saturation in the extractor, so it would thus be expected that most of each would dissolve in the approximately 5.7 ml of mobile phase contained in the extractor. Passage of the mobile phase through the extractor would therefore elute the impurities along with the warfarin enantiomers. As the support was chosen to be unlikely to retain the products, passage of a little under 2 extractor volumes of solvent should suffice to elute all the impurities. This represents a significant concentration of the impurities – roughly 57- fold, given that only 1.7% of the warfarin was eluted (13 mg from 750).

The chromatogram may be compared with the conventional injection of 12 mg of the same sample of warfarin in Figure 3.

An additional set of extraction experiments was run using a second warfarin sample, loaded at 800 mg on 4.1 g of C-1 support. A series of injections each of 11 seconds duration (equal to two extractor volumes) was run and the area of each impurity peak was measured. Figure 4 shows a plot of peak area at 250 nm versus injection number, indicating that most of the impurities were removed after the first injection with only a few low-concentration remnants remaining in the second.

Figure 6. HPLC-MS chromatograms from fractions. W = warfarin, other numbers indicate the mass value of the peaks so annotated. For conditions see the experimental section. Fraction 2 contained no detectable peaks; Fraction 3 was assigned to acetone (the coating solvent). Fractions 7, 8, 11 and 12 contained only warfarin.

from the first injection of 10 seconds (approximately 10 ml) is shown in Figure 1. Multiple impurity peaks were observed. Further injections from the extractor were made after increasing the methanol content of the mobile phase from 15 to 20% to reduce the separation time. Figure 2 shows the 2nd and 3rd injections, of 20 and 30 seconds respectively. The difference between the first and second injections is remarkable and other experiments were run to ensure that the large number of peaks in the first injection did not arise from an external source such as the DAISOGEL 50 micron C-1 support. A sample

of the support loaded into a fresh extractor did not result in eluted peaks. Further, a 600 mg sample of Warfarin was loaded directly (i.e. without coating on a support) into a 50 x 10 mm extractor and a similar series of injections (i.e. 10, 20 and 30 second loadings) were run. Again, the initial injection contained multiple peaks while all subsequent injections contained just the two enantiomers.

The explanation of this at first surprising phenomenon is relatively straightforward. Warfarin is not especially soluble in the supercritical fluid – it was estimated that the solubility in the 20% methanol in CO2

The experiment was repeated, this time while collecting the various peaks eluted from the mixture. A minor adaptation to the standard PIC Solution cyclone collector enabled manual collection in 20 ml scintillation vials. Figure 5 shows the peaks collected. The 21 resulting fractions were analysed by reversed phase HPLC-MS; the resulting chromatograms are shown in Figure 6. Not all UV-active peaks gave useful mass spectra but where acceptable data were obtained the molecular weight(s) associated with the peaks are shown. As the goal of this experiment was not to identify all warfarin impurities but to illustrate that the extraction experiment can be used for the rapid concentration and isolation of impurities, no further attempts were made to analyse the mass spectra or to purify the fractions further. Peaks labelled ‘W’ are warfarin. Fractions 7, 8 and 11 and 12 are not shown; these contain the warfarin enantiomers. Fraction 2 contained

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