The measurement of the β/α anomer composition within amorphous lactose prepared by spray and freeze drying using a simple (1)H-NMR method.

Purpose: Reports of the anomeric composition of amorphous lactose are rare and state a highly variable range of composition (between 0% and 60% w/w β content). We aimed to develop a quantitative measurement by (1)H-NMR of α and β anomer content in amorphous lactose produced by different production methods.

Methods: Amorphous lactose was prepared by spray and freeze drying 10% w/v aqueous solutions of lactose. NMR analysis was performed in DMSO; peak areas of partially resolved doublets at 6.3 and 6.6 ppm were used to calculate % of α and β lactose present. Polarimetery was used to determine optical rotation of lactose solutions.

Results: Observed specific rotation for supplied crystalline alpha lactose monohydrate of 88° recorded in DMSO was constant for the length of a typical NMR experiment (max. 10 min). β/α anomer contents of amorphous lactose measured by (1)H-NMR had standard deviations as low as 0.1% w/w (n = 6). Drying a lactose solution 4 h after its preparation led to almost 35% w/w difference in anomer composition within solid amorphous material compared to samples dried after only 30 min, e.g. for freeze dried samples, β content was 60 ± 0.1% w/w (4 h) and 25 ± 1.0% w/w (30 min). Mutarotation leads to this increase in β anomer concentration in aqueous solution and within the solid amorphous lactose stored at 25°C. e.g. after 56 d storage the β content of freeze dried lactose (30 min solution) increased from 25±1.0% to 50±0.5% w/w.

Conclusion: A simple solution-based (1)H-NMR method for measurement of anomeric composition of lactose has been established. The solution β/α ratio at the time of drying is mirrored in the composition of the resulting solid amorphous material. In order to produce a consistent anomer composition within spray and freeze dried amorphous lactose, the standing time for the feed solution should be greater than 4 h, such that the most dynamic region of the mutarotation profile has been exceeded. If the amorphous material has been formed from a solution that has not been allowed to equilibrate for 4 h, the resulting solid will continue to undergo mutarotation if trace amounts of moisture are present, until the anomeric β/α ratio slowly approaches 1.7.