Malaria is one of the most deadly diseases on earth with an estimated death rate of up to 2.7 million humans per year. The drug primaquine di-phosphate is used for causative treatment of this infection.
Using HPLC equipped with a chiral column and HPLC-MS equipped with a reverse-phase column, we were able to show that the main contaminant of primaquine is the positional isomer quinocide . Using GC, GC-MS we supported previously results and were able to work out the detailed fragmentation map for primaquine and quinocide and to compare the fragmentation . HPLC is the main method in the Pharmacopeias and used in pharmaceutical industries. However, by HPLC, on the columns used , the isomers of primaquine were not adequately separated and therefore it is difficult to quantify the highly toxic contaminant, quinocide. The base line separation is a request in Pharmacopoeia. A novel method for separation of quinocide from primaquine was developed on the HPLC Discovery HS-F5 column resulting in baseline resolution suitable for quantification. Baseline separation was achieved isocratic by varying the buffer proportion in the acetonitrile mobile phase at a flow rate of 1.0 ml/min. The mobile phase composition finally chosen was acetonitrile/20 mM ammonium acetate in distilled water, pH 7.0, 50/50. The analytes were detected at 268 nm with reference at 300 nm.
SFC was also tested as an alternative to HPLC . The Discovery HS-F5 column has advantages in separation on HPLC and SFC over the other columns tested to date. In the experiments with SFC it was possible to get more realistic quantitative results than in HPLC, these results are supporting the previous data of toxicological tests . By using SFC it was also possible to reduce the time for analysis.
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