LC-UV/MS quality analytics of paediatric artemether formulations

Abstract

A highly selective and stability-indicating HPLC-method, combined with appropriate sample preparation steps, is developed for β-artemether assay and profiling of related impurities, including possible degradants, in a complex powder for oral suspension. Following HPLC conditions allowed the required selectivity: a Prevail organic acid (OA) column (250 mm×4.6 mm, 5 μm), flow rate set at 1.5 mL/min combined with a linear gradient (where A=25 mM phosphate buffer (pH 2.5), and B=acetonitrile) from 30% to 75% B in a runtime of 60 min. Quantitative UV-detection was performed at 210 nm. Acetonitrile was applied as extraction solvent for sample preparation. Using acetonitrile-water mixtures as extraction solvent, a compartmental behaviour by a non-solving excipient-bound fraction and an artemether-solubilising free fraction of solvent was demonstrated, making a mobile phase based extraction not a good choice. Method validation showed that the developed HPLC-method is considered to be suitable for its intended regulatory stability-quality characterisation of β-artemether paediatric formulations. Furthermore, LC-MS on references as well as on stability samples was performed allowing identity confirmation of the β-artemether related impurities. MS-fragmentation scheme of β-artemether and its related substances is proposed, explaining the m/z values of the in-source fragments obtained.

Keywords: Artemisinin trioxane derivatives; MS-fragmentation; Paediatric formulations; Polar embedded organic acid column; Related impurities and degradation compounds; Sample preparation.

Fig. 1

β-Artemether recovery in function of percentage acetonitrile in the extraction solvent.

Fig. 2

Percentage acetonitrile found in the test solutions (given as means with 95% confidence intervals) in function of percentage acetonitrile in the extraction solvent.

Fig. 3

Methyl- (○) and propyl- (●) paraben recovery in function of percentage acetonitrile in the extraction solvent.

Fig. 4

Typical chromatograms (UV at 210 nm) showing reference solution (A), placebo solution (B) and spiked placebo solution (C) obtained on the Prevail OA column using the chromatographic conditions as described in the experimental section. Peak identities are as follows: (a) coconut flavour, (b) methyl paraben, (c) propyl paraben, (d) α-dihydroartemisinin, (e) β-dihydroartemisinin, (f) artemisinin, (g) α-artemether, (h) β-artemether, and (i) lumefantrine. Peaks (j) and (k) were identified as lumefantrine-related impurity degradants.

Fig. 5

Mass spectra of β-artemether (A) and artemisinin (B), together with structures allocated to the in-source fragments.

Fig. 6

Proposed fragmentation pattern of artemether, with the rupture of peroxide bridge as proposed in literature (A), as currently proposed (B).

Fig. 7

Proposed fragmentation pattern of artemisinin, with the rupture of peroxide bridge as proposed in literature (A), as currently proposed (B).