Comonomer distribution analysis of poly(3-hydroxybutyrate-<i>co</i>-3-hydroxyhexanoate)s using high-resolution MALDI-TOF mass spectrometry

In practical applications of poly(3-hydroxybutyrate) (PHB) copolyesters, the distribution of comonomer units within the polymer chains (<i>i.e.</i>, comonomer distribution) is an important factor influencing various material properties, alongside the comonomer structure and content. However, analytical methodologies for the characterisation and representation of comonomer distribution in PHB copolyesters remain limited. In the present study, we examined the comonomer distribution in poly(3-hydroxybutyrate-<i>co</i>-3-hydroxyhexanoate) (PHBH) using matrix-assisted laser desorption/ionisation spiral time-of-flight mass spectrometry (MALDI-spiral-TOF-MS). High-molecular-weight PHBH samples were partially methanolysed and subsequently fractionated by size-exclusion chromatography to yield oligomers of suitable molecular sizes for analysis. The high-resolution capabilities of MALDI-spiral-TOF-MS enabled the separation of ion peaks with similar <i>m</i>/<i>z</i> values in the PHBH oligomers and the determination of their accurate masses. The resulting mass spectral data were subsequently subjected to Kendrick mass defect (KMD) analysis to classify ion peaks according to their end-group structures, thereby facilitating the identification of distinct molecular species. Accurate mass measurements enabled the unambiguous determination of the corresponding molecular formulae, end groups, and monomer compositions of the PHBH oligomers. By organising the ion peak data corresponding to PHBH chains of a specific length (<i>e.g.</i>, 21-mer), the distribution of 3-hydroxyhexanoate (3HH) units within the molecular chain was represented as a number distribution plot for that chain length. This analytical approach was also succesfully applied to PHBH film residues subjected to biodegradation in seawater.