Redox speciation of iron, manganese, and copper in cerebrospinal fluid by strong cation exchange chromatography – sector field inductively coupled plasma mass spectrometry

Highlights

• Simultaneous redox speciation method of Fe, Mn, and Cu was designed.

• SCX coupled to ICP-sf-MS was used for the separation and determination of species.

• Method was tested in real cerebrospinal fluid samples.

• Metholloproteins stability was evaluated under separation conditions.

Abstract

A new method of simultaneous redox speciation of iron (II/III), manganese (II/III), and copper (I/II) in cerebrospinal fluid (CSF) has been designed. For the separation of redox species strong cation exchange chromatography (SCX) with isocratic elution was employed. Species were detected using inductively coupled plasma sector field mass spectrometry (ICP-sf-MS), operating at medium resolution. The following parameters were optimized: analytical column, eluent composition and pH, CSF injection volume and dilution factor. Analytical column Dionex IonPac CS5A RFIC 4*250 mm was found to retain and separate species of interest the most effectively under the isocratic elution with a buffer, containing 50 mM ammonium citrate, 7.0 mM pyridine-2,6-dicarboxylic acid at pH = 4.2 and flow rate of 0.8 L min⁻¹. Injection volume of 50 μL with CSF sample dilution of 1/3 (v/v) with the eluent was shown to result in minimal matrix suppression. For species identification, retention time matching with standards was used. The stability of metalloproteins (ferritin, transferrin, and ceruloplasmin) under elution conditions was evaluated. For the quantification of redox species, external calibration was employed. To avoid column contamination, a blank was run after measurement and all quantification values were blank subtracted. For recovery checks, species quantification data was verified against total content of an element, measured by dynamic reaction cell ICP-MS. Recoveries (sum of quantified species vs. total element determinations) were 82.5 ± 22% (Mn), 92 ± 11% (Fe), and 88.7 ± 12% (Cu). The method was tested using 38 real CSF samples. Limits of detection (3σ) for the CSF samples were 0.5 μg L⁻¹, 0.6 μg L⁻¹, and 0.8 μg L⁻¹ for Fe, Mn, and Cu species, respectively. Retention time precision was 1–7.5% (as RSD), whereas peak area RSDs were in the range 5–11%, both depending on the species.

Introduction

Degenerative brain disorders constitute a growing burden for the affected individuals, their families, and the society in general [1]. The same is also true for reliable diagnostic biomarkers at the early stage of the disorders [2]. The major hallmark of the neurodegeneration is deposition of protein aggregates in the brain, consisting of fibers assembled by misfolded proteins with β-sheet conformation [3], [4]. The misfolding of the affected proteins seems to be determined by local factors within the neurons and synapses. Many studies indicate that a variety of environmental factors contribute to the initiation and promotion of neurodegenerative diseases [5], [6], [7]. A number of factors influence protein folding, misfolding and stability, and metal ions are amongst the key effectors [8], [9]. First of all, redox active metals, such as copper, manganese, and iron, play an important role in protein aggregation [10], e.g. by the generation of free radicals via Fenton mechanism [11]. That is why, such transition metals as Cu, Fe, and Mn are of special interest, concerning neurodegenerative states [12].

Chemical speciation is a well-established tool for the study of trace elements biological role and metabolism [5], [13]. The toxicity, bioavailability, and metabolism of the trace elements are well known to be highly dependent on their chemical form. However, the list of the elements with established speciation methodology remains still somewhat limited to arsenic, mercury, and selenium [14], [15], [16], [17], [18], whilst other elements are being paid much less attention. The same is partially true for the objects of the investigation. The researchers are mainly focused on environmental and foodstuff-like matrices [19], [20], [21], whereas speciation studies in clinical samples are less frequent.

Cerebrospinal fluid (CSF) is a well-established media for the human based neurobiological studies related to the metal exposure. This body fluid is an excretion of the choroid plexus in the ventricles of the brain [22]. CSF directly contacts extracellular space of brain parenchyma [23], so depletion of elements or change of element species in the brain is likely to be reflected in this media [24]. Iron, copper, and manganese were previously shown to be well-retained by the blood-brain-barrier, protecting the neuronal tissue from excessive exposure [25]. So, low content of these metals is expected in the CSF and a detection technique for speciation should provide very low detection limits. In the current study, a rapid and simple method of redox state speciation of iron, manganese, and copper in CSF has been designed and tested. Strong cation exchange (SCX) liquid chromatography hyphenated to inductively coupled plasma double focusing sector field mass spectrometry (ICP-sf-MS) was used.