Development and validation of an ultra-performance liquid chromatography with tandem mass spectrometry method for determination of soluble repulsive guidance molecule A in human serum and cerebrospinal fluid

Aim: Repulsive guidance molecule A (RGMa) is upregulated in neurodegenerative diseases. To assess RGMa levels in human serum and cerebrospinal fluid (CSF), a quantification method was developed and validated according to ICH M10 guideline. Methods & results: Sample preparation consisted of immunoprecipitation (IP, only for serum), digestion and purification followed by MS. Conclusion: An UPLC-MS/MS method was established and used to assess normal range of soluble RGMa levels in serum and CSF of healthy controls, and patients with mild cognitive impairment or Alzheimer's disease. The normal range was between 13.0–44.8 ng/ml (CSF) and 9.9–20.9 ng/ml (serum) in healthy controls. In the CSF of patients with mild cognitive impairment and Alzheimer's disease, total soluble RGMa was twofold lower while unchanged in serum. An immunoprecipitation (IP) ultra-performance liquid chromatography (UPLC-MS/MS) method was developed to quantify soluble repulsive guidance molecule A (RGMa) in human serum to support analysis of soluble RGMa in cerebrospinal fluid (CSF) and serum. Samples were prepared using IP for analyte enrichment, tryptic digestion and solid-phase extraction for purification. Measurements were obtained using a UPLC-MS/MS system with CSH C18 columns coupled to a triple quadrupole mass spectrometer operating in positive electrospray ionization. The analytical method has been validated successfully according to the US Food and Drug Administration (FDA) Guidelines within an assay range of 9.8–2500 ng/ml including an anchor point at 4.9 ng/ml in human serum. A fit-for-purpose validation according to the FDA guidelines confirmed the feasibility in CSF as well (assay range, 2.5–200 ng/ml including an anchor point at 1 ng/ml). The method was successfully used to analyze CSF and serum of age-matched patients who had mild cognitive impairment (MCI), patients with Alzheimer's disease (AD) and healthy volunteers from a well-controlled human phase 0 trial. Total soluble RGMa in CSF in healthy volunteers was between 13.0 ng/ml and 44.8 ng/ml (median, 27.5 ng/ml) and in serum being by a factor of two lower between 9.9 ng/ml and 20.9 ng/ml (median, 14.7 ng/ml). In CSF from patients with MCI and those with AD, total soluble RGMa was significantly decreasing to a median of 22.6 and 21.4 ng/ml, respectively. In serum from patients with MCI and those with AD, total soluble RGMa was trendwise increasing to a median of 15.0 and 15.7 ng/ml, respectively. The data points toward a role of total soluble RGMa as a potential neurodegenerative disease biomarker in CSF. However, more data are required to establish biological validation and increase the knowledge about circulating shed forms of RGMa in CSF and serum. Comparing the CSF data with known biomarkers revealed significant correlations between RGMa and Tau in all groups (healthy controls, MCI and AD), and RGMa and pTau-181 in disease states (MCI and AD). Further data analysis is necessary to strengthen our understanding of biomarker correlations.