Development and Validation of a Bioanalytical UHPLC-MS/MS Method Applied to Murine Liver Tissue for the Determination of Indocyanine Green Loaded in H-Ferritin Nanoparticles

Cristina Sottani; Elena Grignani; Danilo Cottica; Serena Mazzucchelli; Marta Sevieri; Arianna Chesi; Fabio Corsi; Sarah Galfrè; Francesco Saverio Robustelli Della Cuna; Enrica Calleri

doi:10.3389/fchem.2021.784123

Development and Validation of a Bioanalytical UHPLC-MS/MS Method Applied to Murine Liver Tissue for the Determination of Indocyanine Green Loaded in H-Ferritin Nanoparticles

Front Chem. 2022 Jan 3:9:784123. doi: 10.3389/fchem.2021.784123. eCollection 2021.

Affiliations

¹ Environmental Research Center, Istituti Clinici Scientifici Maugeri IRCCS, Pavia, Italy.
² Nanomedicine Laboratory, Department of Biomedical and Clinical Sciences "Luigi Sacco", Milano University, Milan, Italy.
³ Breast Unit, Istituti Clinici Scientifici Maugeri IRCCS, Pavia, Italy.
⁴ Department of Drug Sciences, University of Pavia, Pavia, Italy.

Abstract

Indocyanine green (ICG) is one of the most commonly used fluorophores in near-infrared fluorescence-guided techniques. However, the molecule is prone to form aggregates in saline solution with a limited photostability and a moderate fluorescence yield. ICG was thus formulated using protein-based nanoparticles of H-ferritin (HFn) in order to generate a new nanostructure, HFn-ICG. In this study, an ultrahigh performance liquid chromatography-tandem mass spectrometry (UHPLC-MS/MS) system was employed to develop and validate the quantitative analysis of ICG in liver tissue samples from HFn-ICG-treated mice. To precipitate HFn, cold acetone in acidic solution at pH 5.0 was used. The processed liver samples were injected into the UHPLC-MS/MS system for analysis using the positive electrospray ionization mode. Chromatographic separation was achieved on a Waters Acquity UPLC^® HSS T3 Column (1.8 μm, 2.1 × 100 mm) with 0.1% formic acid and acetonitrile as the mobile phase with gradient elution. The selected reaction monitoring transitions of $m / z$ 753 $\to m / z$ 330 and $m / z$ 827 $\to m / z$ 330 were applied for ICG and IR-820 (the internal standard, IS), respectively. The method was selective and linear over a concentration range of 50-1,500 ng/ml. The method was validated for sensitivity, accuracy, precision, extraction recovery, matrix effect, and stability in liver tissue homogenates. ICG extraction recoveries ranged between 85 and 108%. The intra- and inter-day precisions were less than 6.28%. The method was applied to a bio-distribution study to compare the amount of ICG levels from mice treated with HFn-ICG and free ICG. The analyses of the homogenate samples from the two types of treatment showed that the concentration levels of ICG is approximately six-fold higher than those of free ICG (1,411 ± 7.62 ng/ml vs. 235 ± 26.0 ng/ml) at 2 h post injection.

Keywords: FDA validation; UHPLC-MS/MS; biodistribution study; indocynine green; liver tissue.