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Development and validation of a reversed phase liquid chromatographic method with fluorescence detection for the pharmacokinetic study of a new chimeric peptide

Zhang, Xiaoyun, Chen, Ying, Li, Lin, Qiao, Hua, Wang, Rui, Ni, Jingman, You, Jieqiong, Ma, Kaikai, Fan, Chenchen, Fang, Quan
Analytical methods 2016 v.8 no.12 pp. 2620-2627
acetonitrile, blood sampling, computer software, doxorubicin, fluorescence, high performance liquid chromatography, intravenous injection, monitoring, pH, peptides, pharmacokinetics, potassium dihydrogen phosphate, rats, wavelengths
The objective of this research is to develop a sensitive bioanalytical method and investigate the pharmacokinetics of a new chimeric peptide (BN-9) in rats. According to the analysis criteria, a reversed phase high performance liquid chromatography method with fluorescence detection (HPLC-FLD) was established and employed in monitoring the compound in rat plasma using doxorubicin (DOX) as the internal standard (IS). Fluorescent BN-9 and IS were extracted from plasma using dehydrated alcohol. The mobile phase was composed of acetonitrile and potassium dihydrogen phosphate buffer containing 0.05% trifluoroacetic acid (TFA) (pH 7.4; 0.02 M) (30 : 70, v/v) at 0.7 mL min⁻¹. Fluorescence detection was conducted at 490 nm (excitation wavelength) and 520 nm (emission wavelength). The lower limit of quantification (LLOQ) was 0.009 µg mL⁻¹. The intra-batch and inter-batch precisions were less than 8.116%, and the accuracy was within 5.632%. Blood samples were collected after intravenous administration of fluorescent BN-9 at the doses of 5, 10, and 20 mg kg⁻¹ to rats. The main pharmacokinetic parameters were obtained by non-compartmental and compartmental analysis with DAS 2.1.1 software. The t₁/₂α values were 9.584 ± 5.137, 8.548 ± 2.093 and 9.621 ± 0.3224 min. The results showed that the method was successfully applied to investigate the pharmacokinetic profiles of fluorescent BN-9 following intravenous administration to rats.