AUTHOR=Sottani Cristina , Grignani Elena , Cottica Danilo , Mazzucchelli Serena , Sevieri Marta , Chesi Arianna , Corsi Fabio , Galfrè Sarah , Robustelli della Cuna Francesco Saverio , Calleri Enrica 

TITLE=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

JOURNAL=Frontiers in Chemistry

VOLUME=9

YEAR=2022

URL=https://www.frontiersin.org/journals/chemistry/articles/10.3389/fchem.2021.784123

DOI=10.3389/fchem.2021.784123

ISSN=2296-2646

ABSTRACT=<p>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<sup>®</sup> 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 <inline-formula id="inf1"><mml:math id="m1" xmlns:mml="http://www.w3.org/1998/Math/MathML"><mml:mrow><mml:mo> </mml:mo><mml:mrow><mml:mi>m</mml:mi><mml:mo>/</mml:mo><mml:mi>z</mml:mi></mml:mrow></mml:mrow></mml:math></inline-formula> 753 <inline-formula id="inf2"><mml:math id="m2" xmlns:mml="http://www.w3.org/1998/Math/MathML"><mml:mrow><mml:mo>→</mml:mo><mml:mrow><mml:mi>m</mml:mi><mml:mo>/</mml:mo><mml:mi>z</mml:mi></mml:mrow></mml:mrow></mml:math></inline-formula> 330 and <inline-formula id="inf3"><mml:math id="m3" xmlns:mml="http://www.w3.org/1998/Math/MathML"><mml:mrow><mml:mrow><mml:mi>m</mml:mi><mml:mo>/</mml:mo><mml:mi>z</mml:mi></mml:mrow></mml:mrow></mml:math></inline-formula> 827 <inline-formula id="inf4"><mml:math id="m4" xmlns:mml="http://www.w3.org/1998/Math/MathML"><mml:mrow><mml:mo>→</mml:mo><mml:mrow><mml:mi>m</mml:mi><mml:mo>/</mml:mo><mml:mi>z</mml:mi></mml:mrow></mml:mrow></mml:math></inline-formula> 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.</p>