*3.2. Characterization of [(p-AA-CH2-HP)x-co-(HB)y] (Co)oligoester Degradation Products*

The chemical structure of [(p-AA-CH2-HP)x-co-(HB)y] (co)oligoesters, which were subjected to incubation in water after a specific period of time, was determined with the aid of ESI-MS analysis.

Figure 3 shows the negative ESI mass spectrum in a spectral expansion in the range *m*/*z* 600–1100 of the water fractions before (Figure 3a) and after 2 weeks (Figure 3b) of incubation of the (co)oligoesters samples in water at 37 ◦C. The resulting ESI mass spectrum in the case of (co)oligoester consisted of a large number of ions and is much more complex owing to the various combinations of two (p-AA-CH2-HP) and (HB) comonomer units. To simplify the figures and Table 2, the abbreviation for the (p-AA-CH2-HP) comonomer unit was used, that is, (p-AA-CH2-HP)x = Ax. The peaks represented the individual (co)polyester chains at different polymerization degrees and their chemical composition has a tendency to group themselves in clusters. The major mass spacing that occurred between the signals belonging to the neighboring series was equal to 86 Da. These values correspond to the molecular weights of 3-hydroxybutyrate repeating units (86 Da).

**Figure 3.** Spectral expansion in the range *m*/*z* 600–1100 of ESI-mass spectra (performed in negative ion-mode) of water solution of the [(p-AA-CH2-HP)x-co-(HB)y] (co)oligoesters collected before incubation (sample 2) (**a**) and aqua-soluble degradation products collected after hydrolytic degradation (14 days) of the [(p-AA-CH2-HP)x-co-(HB)y] (co)oligoesters at 37 ◦C (**b**).


**Table 2.** Structural assignments of ions present in the enlarged region (*m*/*z* 600–1100) of the electrospray mass spectrometry (ESI-MS) of the degradation products of [(p-AA-CH2-HP)x-co-(HB)y]. Spectra depicted in Figures 3 and 4.

**Figure 4.** ESI-MS2 spectrum (performed in negative-ion mode) of [p-AA-A1/HB3] (co)oligoester ion at *m*/*z* 645 with carboxylic and p-anisate end groups and possessing one p-AA-CH2-HP comonomer unit together with the theoretical fragmentation pathway.

Two main series of ions present in the ESI mass spectra, which appear at *m*/*z* = 151 + (*n* × 86) + 236 and *m*/*z* = 151 + (*n* × 86) + (2 × 236) (Series A and B, Figure 3a and Table 2), correspond to the (co)oligoester chains terminated by carboxylic and p-anisate end groups. The above mentioned (co)oligoester chains contain one (Series A) or two (Series B) (p-AA-CH2-HP) comonomer units arranged in a different combination with HB comonomer units along the oligomer backbone, (Series A) and (Series B), in Figure 3a and Table 2, respectively. In the mass spectra, we can observe a third and less abundant (with lower relative intensity) series of ions Series C (see Figure 3a and Table 2) at *m*/*z* = 151 + (*n* × 86). This series was ascribed to oligo(3-hydroxybutyrate) terminated by carboxyl and p-anisate moiety end groups. Table 2 contains the data with the ascribed structures of the ions presented in the ESI-MS spectra (Figure 3).

Additionally, in the mass spectra shown in Figure 3b, two series of anions, marked as Series D–E, were distinguished. These signals correspond to [(p-AA-CH2-HP)x-co-(HB)y] chains terminated by hydroxyl and carboxylate end groups, which were formed during the hydrolysis process of the (co)oligoester studied. However, the formation of oligo-3-hydroxybutyrate terminated by carboxylate and hydroxyl end groups was not detected in the spectrum. Most probably, the lack of the detection of these signals was due to too low intensity of peaks, below the detection limit. The chemical structures of the identified oligomers (Series A–E) are depicted in Table 2.

A deeper knowledge of the structure of individual (co)oligoester chains was gained with the use of tandem ESI-MS/MS. This technique was also previously applied by us for structural studies of individual molecular ions selected from different types of copolymers and allowing differentiation of the individual molecular chains of the random and diblock copolyesters [27,28].

We applied tandem mass spectrometry (ESI-MS/MS) in order to confirm the structural assignment of the ions present in the obtained ESI-MS spectra. In particular, the acquired data were crucial to verify the distribution of p-AA-CH2-HP and HB comonomers along the [(p-AA-CH2-HP)x-co-(HB)y] (co)oligoester chains. Herein, we show as an example ESI-MS/MS spectrum (in negative ion mode) of the precursor ion at *m*/*z* 645, representing oligomers terminated by carboxyl and p-anisate moiety end groups. The verified ion of interest contained three 3-hydroxybutyrate repeating units and one p-AA-CH2-HP unit distributed randomly in the (co)oligoester chain. The fragment ion at *m*/*z* 493 corresponds to the oligomer formed by the loss of p-AA (152 Da), which can be derived from the terminal group and/or from the pendant group of the p-AA-CH2-HP comonomer unit. The product ion at *m*/*z* 559 represents the oligomer formed by the loss of the 3-hydroxybutyrate unit (crotonic acid; 86 Da) in the oligoester chains. The product ion at *m*/*z* 409 corresponds to the oligomer formed by the loss of the last, in the oligoester chain, p-AA-CH2-HP comonomer unit in the form of 4-methoxybenzoyloxycrotonic acid (236 Da, see Scheme in Figure 4). The ESI-MS/MS spectrum and proposed fragmentation pathway clearly confirm that the p-AA-CH2-HP comonomer units are arranged in a random manner in the oligomer chain.

Fragmentation of the precursor ion at *m*/*z* 645 (Series A, Figure 3 and Table 2) proceeds as a result of random cleavage of ester bonds along the oligomer chain and ester bonds between the chain and of p-anisate pendant group. In the case of solutions containing both acids and oligomers, the intensity of signals in the ESI spectra corresponding to acid molecules is lower than in the case of oligomers [29]. For a quantitative estimation of (co)oligoesters and acid content, based on ESI mass spectra obtained for mixtures, a separate calibration for each type of ingredient is required. Therefore, the release profile of p-anisic acid was determined separately by HPLC (see Section 3.3, *Comparative studies of the release of p-anisic acid from* (p-AA-CH2-HP)n *oligoester and [(p-AA-CH2-HP)x*-co-*(HB)y] (co)oligoesters*)).
