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AAPS PharmSciTech. 2000 March; 1(1): 50–59.
Published online 2015 February 19. doi:  10.1208/pt010106
PMCID: PMC2784833

Orthogonal HPLC methods for quantitating related substances and degradation products of pramlintide

Abstract

Pramlintide is a 37-amino acid peptide that is being evaluated as a drug candidate for treating people with type 1 and insulin-using type 2 diabetes. Two high-performance liquid chromatography (HPLC) methods were developed for quantitating related substance impurities in pramlintide drug substance as well as degradation products of pramlintide formulated for parenteral administration. The methods differ with respect to separation mode and therefore provide orthogonal information concerning related substances and degradation products. One method uses a reverse phase (RP) separation mode, and the other involves a strong cation exchange (SCX) separation. Method performance testing showed that the RP- and SCX-HPLC methods both afford a high degree of selectivity, accuracy, precision, and sensitivity. The limit of quantitation for determining spiked authentic samples of degradation products was shown to be approximately 0.1% (relative to intact pramlintide) for both methods. Relative retention times for known pramlintide degradation products were determined for both the RP- and SCX-HPLC methods, demonstrating the selectivities of the 2 methods as well as the orthogonality of the information. The methods were also shown to be diastereospecific with respect to separating pramlintide from authentic samples of D-isomers at Ala5, Ala8, Ala5-Ala8, and Leu12. The methods did not resolve pramlintide, however, from diastereomers with D-isomers near the C- and N-termini, namely Lys1,Cys2, and Tyr37.

Keywords: Pramlintide, Peptide Analysis, RP-HPLC, SCX-HPLC, Orthogonal Separation, Diastereoselectivity

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Selected References

These references are in PubMed. This may not be the complete list of references from this article.
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