Development and validation of a rapid high‐performance liquid chromatography method for the quantification of exenatide

Abstract

The objective was to develop a simple HPLC method to quantify exenatide--a 39 amino acid residue incretin mimetic used in diabetes therapy. To date, only non-validated, sometimes incomplete, gradient methods have been reported in the literature. Isocratic separation was achieved using a C₄ column and a mixed solvent system, A-B-C (48:45:7, v/v/v; pH* 5.2), where A represents KH₂PO₄ (pH 4.5; 0.1 M) and MeCN (60:40, v/v), B corresponds to NaClO₄ ·H₂O (pH 6.0; 0.2 M) and MeCN (60:40, v/v), and C is water. Exenatide eluted at 3.64 min and the total run time was 6 min. The method was specific and the response was accurate, precise and linear from 0.75 to 25 µg/mL. It was used to quantify exenatide transport across intact and laser-porated porcine skin in vitro as a function of laser fluence [0 (i.e. intact skin), 9 and 15 J/cm², respectively]. Although no permeation was observed using intact skin, cumulative exenatide permeation after 8 h through laser porated skin was 9.6 ± 6.5 and 12.4 ± 6.4 µg/cm² at fluences of 9 and 15 J/cm², respectively. This is the first validated isocratic method for exenatide quantification and it may be of use in quality control analysis and with other biological matrices.

Comparison of ELISA and HPLC-MS methods for the determination of exenatide in biological and biotechnology-based formulation matrices

“…Exenatide is the synthetic form of exendin-4, a 39 amino acid peptide isolated from Gyla monster ( Heloderma suspectum ) saliva [11] with a molecular weight of 4186.6 Da [15] and natural resistance to DPP-IV degradation. It was the first approved GLP-1RA for human clinical application [12], as an adjunctive T2D therapy for patients who have not achieved the optimal glycemic control with oral therapies [16]. Although as a short-acting GLP-1RA with natural resistance to the proteolysis by DPP-IV, exenatide's half-life is 2.4 h, requiring two administrations per day [13], leading to a not simple treatment regimen, compromising the therapeutic adherence [17,18].…”

“…Since exenatide is hydrophilic, the mobile phase used for its elution must be constituted by a small percentage of water, enhancing the interaction between exenatide and the stationary phase and the peak resolution. Regarding Table 3, it is possible to analyze that, generally, researchers use a small proportion of aqueous phase and alkaline pH conditions, since the elution of exenatide with acidic mobile phases is not suitable, due to its cationic behavior [16].…”

A UHPLC-UV Method to Quantify Skin Deposition and Transdermal Permeation of Tizanidine Hydrochloride

“…Absence of interference from endogenous compounds present in the skin was investigated by injecting 10 samples of porcine skin extract (15).…”

The Pharmaceutical Biochemistry Group: Where Pharmaceutical Chemistry Meets Biology and Drug Delivery

“…® ) for minimally invasive drug delivery into and across the skin. [69,[87][88][89][90][91][92] The P. L.E.A.S.E. ® device applies short duration energy pulses (a few µs) at 2.94 µm that excite water molecules in the epidermis and result in their evaporation.…”

“…lidocaine, [89] prednisone [91] and diclofenac [93] ), peptides (e.g. exenatide [88] ) and proteins (e.g. cytochrome c [87] and follicle stimulating hormone).…”