A Rapid and Simple UHPLC-MS/MS Method for Quantification of Plasma Globotriaosylsphingosine (lyso-Gb3)

Fabry disease (FD) is a rare X-linked lysosomal storage disorder caused by α-galactosidase A gene (GLA) mutations, resulting in loss of activity of the lysosomal hydrolase, α-galactosidase A (α-Gal A). As a result, the main glycosphingolipid substrates, globotriaosylceramide (Gb3) and globotriaosylsphingosine (lyso-Gb3), accumulate in plasma, urine, and tissues. Here, we propose a simple, fast, and sensitive method for plasma quantification of lyso-Gb3, the most promising secondary screening target for FD. Assisted protein precipitation with methanol using Phree cartridges was performed as sample pre-treatment and plasma concentrations were measured using UHPLC-MS/MS operating in MRM positive electrospray ionization. Method validation provided excellent results for the whole calibration range (0.25–100 ng/mL). Intra-assay and inter-assay accuracy and precision (CV%) were calculated as &lt;10%. The method was successfully applied to 55 plasma samples obtained from 34 patients with FD, 5 individuals carrying non-relevant polymorphisms of the GLA gene, and 16 healthy controls. Plasma lyso-Gb3 concentrations were larger in both male and female FD groups compared to healthy subjects (<em>p </em>&lt; 0.001). Normal levels of plasma lyso-Gb3 were observed for patients carrying non-relevant mutations of the GLA gene compared to the control group (<em>p </em>= 0.141). Dropping the lower limit of quantification (LLOQ) to 0.25 ng/mL allowed us to set the optimal plasma lyso-Gb3 cut-off value between FD patients and healthy controls at 0.6 ng/mL, with a sensitivity of 97.1%, specificity of 100%, and accuracy of 0.998 expressed by the area under the ROC curve (C.I. 0.992 to 1.000, <em>p</em>-value &lt; 0.001). Based on the results obtained, this method can be a reliable tool for early phenotypic assignment, assessing diagnoses in patients with borderline GalA activity, and confirming non-relevant mutations of the GLA gene.法布里病（Fabry disease, FD）是一种罕见的X连锁溶酶体贮积症，由α-半乳糖苷酶A基因（GLA）突变引发，导致溶酶体水解酶α-半乳糖苷酶A（α-Gal A）活性丧失。由此，其主要糖鞘脂底物神经三己糖酰基鞘氨醇（globotriaosylceramide, Gb3）与溶血神经三己糖酰基鞘氨醇（globotriaosylsphingosine, lyso-Gb3）会在血浆、尿液及组织中蓄积。本研究提出一种简便、快速且灵敏的血浆lyso-Gb3定量检测方法，该物质是法布里病最具应用前景的二次筛查靶点。采用Phree柱辅助甲醇蛋白沉淀法完成样品前处理，并通过在正电喷雾电离多反应监测（multiple reaction monitoring, MRM）模式下运行的超高效液相色谱-串联质谱（ultra-high performance liquid chromatography-tandem mass spectrometry, UHPLC-MS/MS）测定血浆中lyso-Gb3的浓度。方法验证结果显示，全校准范围（0.25~100 ng/mL）内检测性能优异。批内与批间准确度及精密度（变异系数CV%）均小于10%。本方法成功应用于55份血浆样本，样本取自34名法布里病患者、5名携带GLA基因无关多态性的个体以及16名健康对照者。男性与女性法布里病患者组的血浆lyso-Gb3浓度均显著高于健康受试者（*p* < 0.001）。与健康对照组相比，携带GLA基因无关突变的患者血浆lyso-Gb3水平处于正常范围（*p* = 0.141）。将定量下限（lower limit of quantification, LLOQ）降至0.25 ng/mL后，本研究确定法布里病患者与健康对照者的最优血浆lyso-Gb3截断值为0.6 ng/mL，此时灵敏度为97.1%、特异性为100%，以受试者工作特征曲线（Receiver Operating Characteristic, ROC）下面积表示的准确度为0.998（置信区间C.I. 0.992~1.000，*p* < 0.001）。基于上述结果，本方法可作为可靠工具，用于早期表型分型、临界α-Gal A活性患者的诊断评估以及GLA基因无关突变的确认。