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 753 330 and 827 330 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.
Highlights
Indocyanine green (ICG) (Figure 1) is a fluorescent dye which belongs to the family of cyanine dyes
The concentration levels detected from mice treated with free ICG and HFn-ICG decreased over time and detectable levels of ICG were determined up to 24 h
The results indicated a significant difference in the concentration values between ICG quantified by analyzing liver samples from mice treated with HFn-ICG and mice treated with free ICG (1,411 ± 7.62 ng/ml vs. 235 ± 26.0 ng/ ml)
Summary
Indocyanine green (ICG) (Figure 1) is a fluorescent dye which belongs to the family of cyanine dyes. ICG is an amphiphilic molecule, and this characteristic allows its dissolution in both aqueous and lipophilic solvents (Desmettre et al, 2000). The molecule has become decisive as an in vivo imaging agent for different clinical applications (Reinhart et al, 2016; Ferrucci et al, 2018; Shen et al, 2018; Spinoglio et al, 2018; Sevieri et al, 2020). ICG suffers from a high instability as the chemical degradation of this dye depends on the nature of solvents, concentration, temperature, and light exposure. The accumulation of HFn-ICG in different organs of a breast cancer murine model was determined by using IVIS® Lumina II in-vivo Imaging System (Sevieri et al., 2021). For fluorescence pharmacokinetic rates of ICG in mouse liver, within the in vivo imaging techniques, diffusive fluorescence tomography has been used (Zhang et al, 2018)
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