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

<b>Aim:</b> 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. <b>Methods &amp; results:</b> Sample preparation consisted of immunoprecipitation (IP, only for serum), digestion and purification followed by MS. <b>Conclusion:</b> 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.

**研究目的：** 排斥性引导分子A（Repulsive guidance molecule A, RGMa）在神经退行性疾病中表达上调。为评估人血清与脑脊液（cerebrospinal fluid, CSF）中RGMa的水平，本研究依据ICH M10指南开发并验证了一种定量检测方法。
**方法与结果：** 样品前处理包括免疫沉淀（immunoprecipitation, IP，仅用于血清样本）、酶解与纯化，随后进行质谱（mass spectrometry, MS）分析。
**结论：** 本研究建立了超高效液相色谱-串联质谱（ultra-performance liquid chromatography-tandem mass spectrometry, UPLC-MS/MS）检测方法，用于评估健康对照者、轻度认知障碍（mild cognitive impairment, MCI）患者以及阿尔茨海默病（Alzheimer's disease, AD）患者血清与脑脊液中可溶性RGMa的正常参考范围。健康对照者的可溶性RGMa正常参考范围为：脑脊液13.0~44.8 ng/ml，血清9.9~20.9 ng/ml。轻度认知障碍与阿尔茨海默病患者脑脊液中总可溶性RGMa水平较健康对照降低一倍，而血清中该水平无显著变化。本研究开发了一种免疫沉淀-超高效液相色谱-串联质谱（IP-UPLC-MS/MS）方法，用于定量检测人血清中的可溶性排斥性引导分子A（RGMa），以支持脑脊液与血清中可溶性RGMa的分析。样品前处理通过免疫沉淀实现分析物富集，经胰蛋白酶酶解后采用固相萃取进行纯化。检测采用搭载CSH C18色谱柱的UPLC-MS/MS系统，联用正电喷雾电离模式下运行的三重四极杆质谱仪。本分析方法依据美国食品药品监督管理局（U.S. Food and Drug Administration, FDA）指南完成验证，血清检测的线性范围为9.8~2500 ng/ml，包含1个锚定浓度点4.9 ng/ml。依据FDA指南完成的适用性验证也确认了该方法在脑脊液样本中的可行性，脑脊液检测的线性范围为2.5~200 ng/ml，包含1个锚定浓度点1 ng/ml。本方法成功用于分析年龄匹配的轻度认知障碍患者、阿尔茨海默病患者以及来自严格控制的人体0期临床试验的健康志愿者的脑脊液与血清样本。健康志愿者脑脊液中总可溶性RGMa水平为13.0~44.8 ng/ml（中位数27.5 ng/ml），血清中该水平为9.9~20.9 ng/ml（中位数14.7 ng/ml），约为脑脊液的一半。轻度认知障碍与阿尔茨海默病患者脑脊液中总可溶性RGMa水平分别显著降至中位数22.6 ng/ml与21.4 ng/ml。轻度认知障碍与阿尔茨海默病患者血清中总可溶性RGMa水平呈上升趋势，中位数分别为15.0 ng/ml与15.7 ng/ml。本研究数据提示总可溶性RGMa有望成为脑脊液中的神经退行性疾病生物标志物，但仍需更多数据完成生物学验证，并加深对脑脊液与血清中RGMa循环脱落形式的认识。将脑脊液检测数据与已知生物标志物进行对比发现，所有组别（健康对照、轻度认知障碍、阿尔茨海默病）中RGMa与Tau均存在显著相关性，在疾病状态组（轻度认知障碍、阿尔茨海默病）中RGMa与磷酸化Tau-181（pTau-181）也存在显著相关性。尚需进一步数据分析以加深对生物标志物相关性的理解。