2.8. Drug and iron concentration measurements

Major organs from groups A–F were analyzed for drug and for iron (Fe) concentration by mass-spectrometry (MS) and by inductively coupled plasma-mass spectrometry (ICP-MS) respectively. Blood samples were analyzed for drug concentration (by mass spectrometry) but not for iron content. Blood naturally contains iron (bound to hemoglobin) which masks any iron that might be added to blood circulation by administration of nanoparticles to the ear.

Quantification of prednisolone levels in blood plasma and rat tissues by mass-spectrometry was performed by Molecular Mass Spectrometry and Diagnostics (MMSD; Warwick, RI., USA). Following shipping of frozen blood plasma and tissue (liver, kidney, spleen, heart, adrenal glands, and brain) samples to MMSD, all sample preparations for the MS assay were carried out by MMSD. Briefly, at MMSD, tissue samples were first homogenized in 1× PBS. From the plasma and tissue homogenates, sample extraction of prednisolone was performed by protein precipitation with addition of a 1:5 volume of neat acetonitrile with 0.1% formic acid (v/v). The samples were centrifuged at 3000 rpm for 10 min at 10 °C, then the sample supernatants were aliquoted into another 96-well plate for high-performance liquid chromatography (HPLC)-MS/MS assays. The injection volume for electrospray was 10 μL, and the mobile phases used were DI water with acetonitrile and 0.1% formic acid, with a mobile phase flow rate of 300 μL/min.

The effective lower limit of mass spectrometry quantification of prednisolone was 100 pg/mL, and the lower limit of detection was 30 pg/mL. A linear standards curve was generated from 100 pg/mL to 500 ng/mL to assess prednisolone concentration in tissue and blood samples. Quality control samples of low, medium, and high concentrations (5 ng/mL, 50 ng/mL, and 500 ng/mL respectively) were run in between every 15 samples throughout the analysis. The quality control sample variability indicated that the sample-to-sample variation was within an acceptable range of 4.5% to 7.2%.

The nanoparticles contain iron-oxide cores to make them magnetic. Two and thirty days after treatment, iron concentrations in tissue samples were quantified by inductively coupled plasma-mass spectroscopy (ICP-MS), following the measurement protocol provided by PerkinElmer and in line with prior published protocols (Kut et al., 2012; Nixon et al., 2000; Thomas, 2013; USDA Food Safety and Inspection Service, 2013; Wegst-Uhrich et al., 2015). Tissue samples were dried in an oven at 93 °C for 4–6 h, and the dried samples were weighed. Then the dried tissue samples were acid digested with 70% nitric acid for 24 to 48 h at 37 °C. In order to achieve complete digestion, 30% hydrogen peroxide (1:1 with nitric acid v/v) was added to fatty tissues such as brain, liver and kidneys. Digested samples were then diluted with 18.2 Ω UV ultra-purified water, and filtered with 0.22 μm PVDF Millex-GV 13 mm filters (Millipore, Cork, Ireland) for the ICP-MS assays.

The tissue concentrations of iron were analyzed by a NexIon300D ICP-MS (PerkinElmer, USA) instrument at the Molecular Characteristics Analysis Complex at the University of Maryland Baltimore County (Baltimore, MD). Inductively coupled plasma mass spectrometry is an analytical technique used for elemental determinations. Compared to other methods such as ICP-AES (ICP with atomic emission spectroscopy) and GFAAS (graphite furnace atomic absorption), ICP-MS has both a lower limit of detection and a higher maximal detection limit, as well as an ability to analyze a wider variety of elements (Thomas, 2013). Using ICP-MS, the amount of iron in tissue samples was determined using a standards curve generated by known amounts of iron in certified iron standards (Fluka/Sigma-Aldrich, Buchs, Switzerland). The iron limit of detection was 10 ng (iron)/g (tissue). Iron concentration was measured in major organs (brain, heart, liver, spleen, adrenal glands, and kidneys) two and thirty days after magnetic administration to the ear.