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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.


MS/MS spectra of observed peaks in Fig. 6 at m/z 1079.
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Fig7: MS/MS spectra of observed peaks in Fig. 6 at m/z 1079.

Mentions: Figure 7 shows the LC–MS/MS analysis of the peaks observed in the EIC at m/z 1079. MS/MS revealed complete amino acid sequence information by a comprehensive series of b-ions which carry the N-terminus. The MS/MS of peak 1 (in Fig. 6) shows that all b ions up to b7 can be attributed to the sequence of the native peptide, indicating that acylation had occurred on the last amino acid (position 8, Fig. 1). The MS/MS results however cannot distinguish whether the primary or the secondary hydroxyl group of this position 8 group has been acylated. However, no acylation was observed on the secondary hydroxyl of threonine at position 6 and therefore it is concluded that the primary hydroxyl of position 8 is susceptible for acylation. In the second peak of the chromatogram in Fig. 6, the b4 ion was found unaffected while b5 incremented by 58 Da, indicating that lysine (position 5) has been acylated. In the last peak (number 3), all b-ions where shifted by +58 Da, demonstrating that the amine of the N-terminus was acylated. The ion at m/z 1109 of octreotide released from PLGHMGA microspheres corresponds with addition of one HMGA unit to the native peptide (Table II). The extracted-ion chromatogram of this ion also showed 3 peaks pointing to the same sites of modification as observed for the octreotide-GA adduct (FigureS1 & S2). Figure S3 shows four peaks for the extracted ion chromatogram at m/z 1093, which corresponds with addition of one lactic acid unit to octreotide. The MS/MS spectrum (Figure S4) of the first peak indicates the addition of the LA unit on the primary hydroxyl of the terminal amino acid, whereas the second peak is ascribed to addition on Lys, while peaks 3 & 4 have the exact same fragmentation pattern both indicating the addition of LA on the N-terminus. We presume that this is the result of the formation of diastereoisomers because the polymer contained both d- and l-lactic acid units. The same analysis was performed for all the observed ions that are mentioned in Table II for the peptide released from the other two microspheres (PLGA and PLHMGA);the distributions of acylation adducts are summarized in Fig. 8. One can conclude that the N-terminus of octreotide is the most susceptible site for acylation, with the acylation by glycolic acid (if present in the copolymer) being the most abundant.Fig. 7


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)

MS/MS spectra of observed peaks in Fig. 6 at m/z 1079.
© Copyright Policy - OpenAccess
Related In: Results  -  Collection

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

Fig7: MS/MS spectra of observed peaks in Fig. 6 at m/z 1079.
Mentions: Figure 7 shows the LC–MS/MS analysis of the peaks observed in the EIC at m/z 1079. MS/MS revealed complete amino acid sequence information by a comprehensive series of b-ions which carry the N-terminus. The MS/MS of peak 1 (in Fig. 6) shows that all b ions up to b7 can be attributed to the sequence of the native peptide, indicating that acylation had occurred on the last amino acid (position 8, Fig. 1). The MS/MS results however cannot distinguish whether the primary or the secondary hydroxyl group of this position 8 group has been acylated. However, no acylation was observed on the secondary hydroxyl of threonine at position 6 and therefore it is concluded that the primary hydroxyl of position 8 is susceptible for acylation. In the second peak of the chromatogram in Fig. 6, the b4 ion was found unaffected while b5 incremented by 58 Da, indicating that lysine (position 5) has been acylated. In the last peak (number 3), all b-ions where shifted by +58 Da, demonstrating that the amine of the N-terminus was acylated. The ion at m/z 1109 of octreotide released from PLGHMGA microspheres corresponds with addition of one HMGA unit to the native peptide (Table II). The extracted-ion chromatogram of this ion also showed 3 peaks pointing to the same sites of modification as observed for the octreotide-GA adduct (FigureS1 & S2). Figure S3 shows four peaks for the extracted ion chromatogram at m/z 1093, which corresponds with addition of one lactic acid unit to octreotide. The MS/MS spectrum (Figure S4) of the first peak indicates the addition of the LA unit on the primary hydroxyl of the terminal amino acid, whereas the second peak is ascribed to addition on Lys, while peaks 3 & 4 have the exact same fragmentation pattern both indicating the addition of LA on the N-terminus. We presume that this is the result of the formation of diastereoisomers because the polymer contained both d- and l-lactic acid units. The same analysis was performed for all the observed ions that are mentioned in Table II for the peptide released from the other two microspheres (PLGA and PLHMGA);the distributions of acylation adducts are summarized in Fig. 8. One can conclude that the N-terminus of octreotide is the most susceptible site for acylation, with the acylation by glycolic acid (if present in the copolymer) being the most abundant.Fig. 7

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.