Development and validation of a stability-indicating LC–UV method for the determination of pantethine and its degradation product based on a forced degradation study

• Degradation products of pantethine were identified by LC–MS/MS analysis.
• Hydrolytic and oxidative degradation pathways of pantethine were elucidated.
• The LC–UV method was validated and applied for pantethine and its degradation product assays.
• The LC–UV method was applied for determination of preservant system.
• The LC–UV method was applied for analysis of real samples in stability studies.

Pantethine (d-bis-(N-pantothenyl-β-aminoethyl)-disulfide, PAN), the stable disulfide form of pantetheine, has beneficial effects in vascular diseases being able to decrease the hyperlipidaemia, moderate the platelet function and prevent the lipid peroxidation. Furthermore, recent studies suggested that PAN may be an effective therapeutic agent for cerebral malaria and, possibly, for neurodegenerative processes. Interestingly, in the literature, there were no data dealing with the chemical stability and the analytical aspects of PAN. Hence, in the present work the chemical stability of PAN was for the first time established through a forced degradation study followed by liquid chromatography tandem mass spectrometry investigation showing the formation of three degradation products of PAN (PD1, PD2 and POx) arising from hydrolytic, thermal and oxidative stresses. Based on these data a stability-indicating LC–UV method for simultaneous estimation of PAN, and its most relevant degradation product (PD1) was developed and validated; moreover the method allowed also the separation and the quantification of the preservative system, constituted by a paraben mixture. The method showed linearity for PAN (0.4–1.2 mg mL−1), MHB, PHB (0.4–1.2 μg mL−1) and PD1 (2.5–100 μg mL−1); the precision, determined in terms of intra-day and inter-day precision, expressed as RSDs, were in the ranges 0.4–1.2 and 0.7–1.4, respectively. The method demonstrated to be accurate and robust; indeed the average recoveries were 100.2, 99.9, and 100.0% for PAN, MHB and PHB, respectively, and 99.9% for PD1. By applying small variations of the mobile phase composition, counter-ion concentration and pH the separation of analytes was not affected. Finally, the applicability of this method was evaluated analyzing the available commercial forms at release as well as during stability studies.

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