Key considerations for phosphatidylserine quantification by ³¹P NMR in pharmaceutical analysis

Phosphatidylserine (PS) is a phospholipid essential for cell membrane composition and with high concentrations in the brain. PS is widely used as a dietary supplement due to its neuroprotective effects and the benefits shown to patients with Central Nervous System (CNS) disorders. This work aimed to identify critical aspects in the development of a quantitative NMR (qNMR) method using quantitative ³¹P NMR (qPNMR) with triphenyl phosphate (TPP) as an internal calibrant (IC) for the analysis of PS samples. The findings indicate several pivotal aspects in the method development: i) sample preparation is a critical step for successful qNMR analysis; the addition of EDTA 0.2 M (pH 7.2-7.5, adjusted with Cs₂CO₃) enhances the lineshape and, thus, the overall resolution of the spectra; ii) the molecular weight of the analyte significantly impacts the analytical outcomes; and iii) post-acquisition processing conditions, including window functions and integration criteria, have slight but noticeable effects on the quantitative outcomes and should, therefore, be considered an integral part of any convention method for PS quantitation. To overcome this latter aspect, peak fitting was found to be an effective approach for reducing operator bias, especially in integration, and is viable due to the essential near-singlet nature of the ³¹P NMR peak patterns for each individual chemical species.