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Identification and Assessment of Octreotide Acylation in Polyester Microspheres by LC-MS/MS.

Shirangi M, Hennink WE, Somsen GW, van Nostrum CF - Pharm. Res. (2015)

Bottom Line: Release profiles of octreotide from hydrophilic microspheres were compared with that of PLGA microspheres.Nucleophilic attack of the peptide can also occur to the carbamate bond presented in (PC-PEG-PC)-(PL) since 1,4-butanediisocyanate was used as the chain extender.LC-ITMS provided detailed structural information of octreotide modifications via mass analysis of ion fragments.

View Article: PubMed Central - PubMed

Affiliation: Department of Pharmaceutics, Utrecht Institute for Pharmaceutical Sciences, Utrecht University, Utrecht, The Netherlands.

ABSTRACT

Purpose: Polyesters with hydrophilic domains, i.e., poly(D,L-lactic-co-glycolic-co-hydroxymethyl glycolic acid) (PLGHMGA) and a multiblock copolymer of poly(ε-caprolactone)-PEG-poly(ε-caprolactone) and poly(L-lactide) ((PC-PEG-PC)-(PL)) are expected to cause less acylation of encapsulated peptides than fully hydrophobic matrices. Our purpose is to assess the extent and sites of acylation of octreotide loaded in microspheres using tandem mass spectrometry analysis.

Methods: Octreotide loaded microspheres were prepared by a double emulsion solvent evaporation technique. Release profiles of octreotide from hydrophilic microspheres were compared with that of PLGA microspheres. To scrutinize the structural information and localize the actual modification site(s) of octreotide, liquid chromatography ion-trap mass spectrometry (LC-ITMS) was performed on the acylated adducts.

Results: Hydrophilic microspheres showed less acylated adducts in comparison with PLGA microspheres. LC-MS/MS showed that besides the N-terminus and primary amine of lysine, the primary hydroxyl of the end group of octreotide was also subjected to acylation. Nucleophilic attack of the peptide can also occur to the carbamate bond presented in (PC-PEG-PC)-(PL) since 1,4-butanediisocyanate was used as the chain extender.

Conclusions: Hydrophilic polyesters are promising systems for controlled release of peptide because substantially less acylation occurs in microspheres based on these polymers. LC-ITMS provided detailed structural information of octreotide modifications via mass analysis of ion fragments.

No MeSH data available.


Proposed mechanism of reaction between peptide and carbamate group in (PC-PEG-PC)-(PL) multiblock copolymer.
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Fig9: Proposed mechanism of reaction between peptide and carbamate group in (PC-PEG-PC)-(PL) multiblock copolymer.

Mentions: For octreotide released from the (PC-PEG-PC)-(PL), two main peaks were observed in LC–MS with m/z values that were 72 and 212 Da higher than observed for native octreotide (Table III). The peak with m/z 1091 is ascribed to LA addition to octreotide. Since 1,4-butanediisocyanate has been used as the chain extender connecting the PC-PEG-PC with poly(lactic acid) to form the poly((ε-caprolactone-b-PEG-b-ε-caprolactone)-b-l-lactide)) multiblock copolymer, we propose that nucleophiles of the peptide can react with the carbonyl group of the carbamate bond (Fig. 9). Consequently, after full hydrolysis of the remaining ester groups, a butanediisocyanate bound by a carbamate bond to one lactic acid unit remains coupled to the peptide. Indeed, the molecular weight of this group is 212 Da and explains the presence of the m/z 1231 ion. It should be noted that for quantitative measurement of the acylated products of the peptide released from (PC-PEG-PC)-(PL) microspheres (Figs. 4d & 5d), fluorescence was used for detection in order to avoid the overestimation of acylated adducts at UV 210 because of the absorbance by the carbamate bond at this wavelength (34).Table III


Identification and Assessment of Octreotide Acylation in Polyester Microspheres by LC-MS/MS.

Shirangi M, Hennink WE, Somsen GW, van Nostrum CF - Pharm. Res. (2015)

Proposed mechanism of reaction between peptide and carbamate group in (PC-PEG-PC)-(PL) multiblock copolymer.
© Copyright Policy - OpenAccess
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC4526596&req=5

Fig9: Proposed mechanism of reaction between peptide and carbamate group in (PC-PEG-PC)-(PL) multiblock copolymer.
Mentions: For octreotide released from the (PC-PEG-PC)-(PL), two main peaks were observed in LC–MS with m/z values that were 72 and 212 Da higher than observed for native octreotide (Table III). The peak with m/z 1091 is ascribed to LA addition to octreotide. Since 1,4-butanediisocyanate has been used as the chain extender connecting the PC-PEG-PC with poly(lactic acid) to form the poly((ε-caprolactone-b-PEG-b-ε-caprolactone)-b-l-lactide)) multiblock copolymer, we propose that nucleophiles of the peptide can react with the carbonyl group of the carbamate bond (Fig. 9). Consequently, after full hydrolysis of the remaining ester groups, a butanediisocyanate bound by a carbamate bond to one lactic acid unit remains coupled to the peptide. Indeed, the molecular weight of this group is 212 Da and explains the presence of the m/z 1231 ion. It should be noted that for quantitative measurement of the acylated products of the peptide released from (PC-PEG-PC)-(PL) microspheres (Figs. 4d & 5d), fluorescence was used for detection in order to avoid the overestimation of acylated adducts at UV 210 because of the absorbance by the carbamate bond at this wavelength (34).Table III

Bottom Line: Release profiles of octreotide from hydrophilic microspheres were compared with that of PLGA microspheres.Nucleophilic attack of the peptide can also occur to the carbamate bond presented in (PC-PEG-PC)-(PL) since 1,4-butanediisocyanate was used as the chain extender.LC-ITMS provided detailed structural information of octreotide modifications via mass analysis of ion fragments.

View Article: PubMed Central - PubMed

Affiliation: Department of Pharmaceutics, Utrecht Institute for Pharmaceutical Sciences, Utrecht University, Utrecht, The Netherlands.

ABSTRACT

Purpose: Polyesters with hydrophilic domains, i.e., poly(D,L-lactic-co-glycolic-co-hydroxymethyl glycolic acid) (PLGHMGA) and a multiblock copolymer of poly(ε-caprolactone)-PEG-poly(ε-caprolactone) and poly(L-lactide) ((PC-PEG-PC)-(PL)) are expected to cause less acylation of encapsulated peptides than fully hydrophobic matrices. Our purpose is to assess the extent and sites of acylation of octreotide loaded in microspheres using tandem mass spectrometry analysis.

Methods: Octreotide loaded microspheres were prepared by a double emulsion solvent evaporation technique. Release profiles of octreotide from hydrophilic microspheres were compared with that of PLGA microspheres. To scrutinize the structural information and localize the actual modification site(s) of octreotide, liquid chromatography ion-trap mass spectrometry (LC-ITMS) was performed on the acylated adducts.

Results: Hydrophilic microspheres showed less acylated adducts in comparison with PLGA microspheres. LC-MS/MS showed that besides the N-terminus and primary amine of lysine, the primary hydroxyl of the end group of octreotide was also subjected to acylation. Nucleophilic attack of the peptide can also occur to the carbamate bond presented in (PC-PEG-PC)-(PL) since 1,4-butanediisocyanate was used as the chain extender.

Conclusions: Hydrophilic polyesters are promising systems for controlled release of peptide because substantially less acylation occurs in microspheres based on these polymers. LC-ITMS provided detailed structural information of octreotide modifications via mass analysis of ion fragments.

No MeSH data available.