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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.


UPLC of octreotide released after 45 days in PBS pH 7.4 at 37°C from (a) PLGA, (b) PLGHMGA, (c) PLHMGA and (d) (PC-PEG-PC)-(PL) (the latter after 28 days).
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Fig4: UPLC of octreotide released after 45 days in PBS pH 7.4 at 37°C from (a) PLGA, (b) PLGHMGA, (c) PLHMGA and (d) (PC-PEG-PC)-(PL) (the latter after 28 days).

Mentions: UPLC-UV chromatograms of octreotide released after 45 days from PLGA, PLGHMGA and PLHMGA, respectively, and from (PC-PEG-PC)-(PL) after 28 days, are shown in Fig. 4. The main peak eluting after approx. 2.5 min corresponds to native octreotide, while the extra peaks with longer retention times are originating from acylated octreotide adducts (see LC–MS analysis). It is remarkable that the extent of acylated adduct is significantly more pronounced for octreotide released from PLGA microspheres than that released from the other microspheres. It should be noted that only native octreotide was detected when the microspheres were dissolved immediately after preparation, demonstrating that the manufacturing process did not cause the acylation of the peptide. In line herewith, it has been observed that acylation of peptides occurs during the in vitro release from PLGA microspheres when the polymer starts to degrade (14). Peptide acylation is catalyzed by the low pH that is generated inside degrading PLGA microspheres due to the accumulation of acid degradation products, i.e., glycolic and lactic acid and soluble oligomers thereof (9,14,29,30).Fig. 4


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)

UPLC of octreotide released after 45 days in PBS pH 7.4 at 37°C from (a) PLGA, (b) PLGHMGA, (c) PLHMGA and (d) (PC-PEG-PC)-(PL) (the latter after 28 days).
© Copyright Policy - OpenAccess
Related In: Results  -  Collection

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Fig4: UPLC of octreotide released after 45 days in PBS pH 7.4 at 37°C from (a) PLGA, (b) PLGHMGA, (c) PLHMGA and (d) (PC-PEG-PC)-(PL) (the latter after 28 days).
Mentions: UPLC-UV chromatograms of octreotide released after 45 days from PLGA, PLGHMGA and PLHMGA, respectively, and from (PC-PEG-PC)-(PL) after 28 days, are shown in Fig. 4. The main peak eluting after approx. 2.5 min corresponds to native octreotide, while the extra peaks with longer retention times are originating from acylated octreotide adducts (see LC–MS analysis). It is remarkable that the extent of acylated adduct is significantly more pronounced for octreotide released from PLGA microspheres than that released from the other microspheres. It should be noted that only native octreotide was detected when the microspheres were dissolved immediately after preparation, demonstrating that the manufacturing process did not cause the acylation of the peptide. In line herewith, it has been observed that acylation of peptides occurs during the in vitro release from PLGA microspheres when the polymer starts to degrade (14). Peptide acylation is catalyzed by the low pH that is generated inside degrading PLGA microspheres due to the accumulation of acid degradation products, i.e., glycolic and lactic acid and soluble oligomers thereof (9,14,29,30).Fig. 4

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.