RAPID COMMUNICATIONS IN MASS SPECTROMETRY, VOL. zyxwvu 6, 702-706 (1992) zyxwv Sequence Distribution of P-Hydroxyalkanoate Units in Bacterial Copolyesters Determined by Desorption Chemical Ionization Mass Spectrometry Rosaria Abate, Domenico Garozzo and Roberto Rapisardi Istituto per la Chimica e la Tecnologia dei Materiali Polirnerici, Consiglio Nazionale delle Ricerche, Viale A. Doria, 6, 95125 Catania, Italy Albert0 Ballistreri and Giorgio Montaudo* Dipartimento di Scienze Chimiche, Universita di Catania, Viale A. Doria, 6, 95125 Catania, Italy The sequence distributions of zyxwvut poly(P-hydroxyalkanoate) copolymers were determined by analyzing oligomers obtained by the pyrolysis direct-chemical-ionization (DCI) mass spectrometry technique. Oligomers up to nonamers were identified and the comparison between the experimental and calculated peak intensities makes it possible to calculate repeating unit compositions and sequence distributions. Comparison with other earlier methods are given. Poly(6-hydroxyalkanoates) (PHAs) are optically active polyesters that serve as storage material for a wide variety of bacteria.' These biopolymers, which are produced in the form of small granules within the cell, have the general structure &-IH-CH~-CJ in which R is an n-alkyl group varying in size from CH, to C12H25 and higher, depending on the bacterium. The structural characterization of PHA copolymers that contain P-hydroxybutyrate (HB) and P-hydroxy- valerate (HV) has attracted much attention recently2-I4 because they are now available on the market and are becoming important materials in biotechnology zyxwvut . They are biocompatible, biodegradable and possess good mechanical The sequence distribution of P(HB-co-HV) copoly- mers has been deduced from dyad and triad analysis I3C NMR spectrometry, and it has been concluded that some of the bacterially synthesized P(HB-co-HV) sam- ples are random copolymers of HB and HV units, but that others are mixtures of P(HB-co-HV) random copolymers with different c~mpositions.~ However, NMR can hardly discern beyond the triad level, and the distinction between a simple copolymer and a mixture of copoloymers is difficult zyxwvuts .4 Furthermore, the compo- sitions and sequence distributions of the repeating units in PHAs with long alkyl pendant groups cannot be determined by NMR spectrometry because the chemi- cal shifts of the carbon atoms in the units are essentially identical.' The application of mass spectrometry is not limited by the size of the repeating units, since large oligomers can be identified. We have described how such natural copolyesters can be analyzed for composition and sequence distribu- tion by fast-atom bombardment mass spectrometry (FAB-MS) of the oligomers formed by partial methanoly~is~,~ and by partial pyrolysis'"-'3 of the co- Author to whom correspondence should be addressed. polymers. It was found8-14that ion intensities in FAB mass spectra of PHAs reflect the relative amounts of the oligomers produced in the partial degradation, and this permits the determination of the composition and microstructure of the copolymers. Thus, FAB-MS was used to determine the structure of these bacterial copolyesters. We have derived their composition and microstruc- ture by using chain ~tatistics,~-'~ a novel application, since chain statistical analysis of copolymers had no precedent in the literature, as far as mass spectrometry was concerned. " In this report we describe a method for the structural analysis of poly(P-hydroxyalkanoate) copolymers, based on the analysis of the pyrolysis products obtained by fast heating with the standard direct-chemical- ionization (DCI) tip. DCI mass spectra of synthetic and naturally occur- ring polymers have been reported,"-'* and it has been found that the DCI technique permits very rapid heat- ing rates and provides a large surface area for dispers- ing the sample within the ionizing plasma. In our experiments rapid heating of the DCJ tip produces pyrolysis zyxwv of the polymers, followed by the desorption of the products. We illustrate two representative examples showing that the negative-ion DCI spectra of the poly-6- hydroxyalkanoates allow the identification of oligomers up to the heptamer or nonamer level. The comparison between the experimental and calculated peak intensit- ies corresponding to higher oligomers makes it possible to determine the composition of the copolymers. EXPERIMENTAL zyxw Materials. Sample 1 is a copolyester with a monomeric composition of P-hydroxyvalerate (HV)/P- hydroxyheptanoate (HH)lP-hydroxynonanoate (HN)/ 6-hydroxyundecanoate (HU) of 2/31/65/2 mol% (MW 120 000). It was obtained in the laboratory of Professor R. W. Lenz, University of Massachusetts, Amherst, MA, USA and it had previously been analyzed by 095 1-4 198/92/ 1 10702-05 $07.50 zyxwvutsr 0 1992 by John Wiley & Sons, Ltd Received 24 August 1992 Accepted 15 September zy I992