Purpose: Iron deficiency anemia is the most common nutritional deficiency worldwide, affecting nearly 1 billion people. As of October 2017, there are six brand (Ferinject, Feraheme, Venofer, Ferrlecit, Dexferrum, InFed) and one generic (Sodium Ferric Gluconate, SFG) referencing Ferrlecit) FDA-approved intravenous (IV) iron products on the U.S. market for the treatment of iron deficiency anemia. These products are colloidal nanoparticles composed of a polynuclear iron(III)-(oxy)hydroxide core, stabilized by carbohydrate ligands. Under iron overload conditions with saturated transferrin, labile iron will be taken up by the cell via non-iron specific pathways and promote the production of reactive oxygen species which can damage proteins, DNA and lipids. A key gap in our understanding is how iron is released from iron nanoparticle drugs. Current methods do not allow for the iron speciation of the nanoparticle drug and the simultaneous quantification of all the iron species in the plasma. The objective of this study was to develop novel bioanalytical methods using inductively coupled plasma-mass spectrometry (ICP-MS) and liquid chromatography (LC)-coupled ICP-MS for the simultaneous identification and quantification of all iron species in support of the ongoing comparative pharmacokinetic bioequivalence studies comparing brand and generic SFG complex products. The iron species include total iron (TI), transferrin-bound iron (TBI), protein-bound iron (PBI), labile iron (LI), and drug-bound iron (DBI) (i.e. iron in SFG complex products) in human plasma in their native forms.
Methods: To quantify TI, the human plasma spiked with SFG was diluted with milliQ water to achieve an iron concentration range of 0.2-40 ppm in 15 mL meta-free centrifuge tubes. 500 mL nitric acid was added to 1000 mL samples incubated at 80oC for 18 hr to release all iron ions, which are then quantified by the ICP-MS analysis. To quantify DBI, the human plasma spiked with SFG was diluted with 10 mM Tris base buffer at pH 7.4 to achieve an iron concentration range of 0.3-50 ppm. To quantify TBI, LI, and PBI, TBI with crystallized human holo-transferrin (Sigma Aldrich) at an iron concentration range of 10-1400 ppb and internal iron standards were prepared. Individual iron species including TBI, PBI (e.g. albumin, ferritin), LI, and DBI, were separated by LC on an Agilent 1260 Infinity HPLC equipped with a pump, an auto-sampler, a column compartment, and a multiple wavelength detector. TI and LC-separated individual iron species were then centrifuged (4000 x g, 10 min, 25 oC) to remove any precipitated proteins before the ICP-MS analysis, using an Agilent 7700x ICP-MS (Agilent Technologies, Santa Clara, CA) based on the following parameters: RF power of 1550 W, argon carrier gas flow of 0.99 L/min, helium gas flow of 4.3 mL/min, octopole RF of 190 V, and OctP bias of -18 V.
Results: The iron species TI (0.2-40 ppm iron), TBI/PBI/LI (10-1400 ppb iron) and DBI (0.3-50 ppm iron) in human plasma were quantified using the ICP-MS and LC-ICP-MS analyses. The table below summarizes the coefficients of variation (CV) of percent recovery, inter-batch precision, within-run precision, free-thaw stability, short-term stability (25oC, 24 hr), long-term stability (-20oC with different duration), and post-preparative benchtop and auto-sampler stability (different conditions). It is noteworthy that the LI component of plasma is stable up to only one freeze-thaw cycle.
Conclusion: As shown in the table above, the observed CVs of percent recovery, inter-batch precision, within-run precision and all stability conditions for all iron species are considered acceptable because the values are within the recommended limit (i.e., <20% deviation from the LLOQ and <15% deviation for all the other samples). Due to the highly unstable LI, certain preparatory procedures need to be adjusted, e.g., preparing plasma samples at 0oC (ice bath) within one hour after thawing the plasma samples for only one freeze-thaw cycle and maintaining the HPLC auto-sampler at 4oC. The developed and validated bioanalytical methods of all iron species in human plasma will be used in support of the upcoming two-way crossover in vivo bioequivalence pharmacokinetic study comparing all iron species levels between brand and generic SFG complex drug products in healthy subjects.
Sergei Alexishin– University of Maryland School of Pharmacy
Joel Brandis– Doctoral Candidate, University of Maryland Baltimore, Baltimore, Maryland
Anne Williams– University of Maryland School of Pharmacy
Dajun Sun– US Food and Drug Administration, Silver Spring, Maryland
Nan Zheng– U.S. Food and Drug Administration (FDA)
Wenlei Jiang– U.S. Food and Drug Administration (FDA), Maryland
James Polli– Professor and Ralph F. Shangraw/Noxell Endowed Chair in Industrial Pharmacy and Pharmaceutics, University of Maryland School of Pharmacy, Maryland
Maureen Kane– University of Maryland School of Pharmacy
Sarah Michel– Professor, University of Maryland School of Pharmacy