A comprehensive approach for detection of biotin deficiency from dried blood spot samples using liquid chromatography-mass spectrometry

<b>Aim:</b> The aim of the present study is to develop a liquid chromatography-mass spectrometry method to measure two important biomarkers of biotin deficiency from dried blood spot samples for effective management of the disorder. <b>Materials &amp; methods:</b> The method was developed on a liquid chromatography-mass spectrometry system using pentafluorophenyl column employing a mobile phase composition of methanol and water in the isocratic mode. A full validation of the method was performed as per relevant guidelines. <b>Results &amp; conclusion:</b> Correlation between the results of dried blood spot and plasma method was evaluated to determine the interconvertibility of the method. The developed method was successfully applied for establishing the reference ranges for these biomarkers in the population of Udupi, a coastal district of South India. Biotin deficiency can lead to many complications such as impaired growth, compromised immune function, depression, myalgia and may even lead to death. The disorder can be managed by supplementation of biotin. Early detection is crucial in managing biotin deficiency. In this paper we describe a comprehensive method for the early detection of biotin deficiency. The method employs the use of minimally invasive blood sampling such as dried blood spot that is suitable for vulnerable neonatal population. The LC-MS method targets the simultaneous determination of 3-hydroxyisovaleric acid (3-HIV) and 3-hydroxyisovaleryl carnitine (3-HIC) in dried blood spots (DBS), providing a comprehensive analysis for diagnosing and preventing biotin deficiency in newborns and infants. Simple, selective, rapid, specific and highly sensitive. The method was validated for accuracy, precision, recovery, matrix effect, hematocrit effect, stability and blood volume/punch position effects. High linearity (R2 &gt;0.99), good sensitivity (LLOQ 0.088 μM and 0.040 μM for 3-HIV and 3-HIC respectively). Recovery of QC samples was within acceptable limits for both analytes. Strong correlation and minimal bias were found between DBS and plasma measurements. Applied successfully for establishing reference ranges in the population of Udupi, South India. 3-HIV (0.06–18.59 μM) and 3-HIC (0.01–0.49 μM) in Udupi population. The newly developed method is a promising tool for confirmatory diagnosis of biotin deficiency in a clinical setting.

**研究目的：** 本研究旨在开发一种液相色谱-质谱（liquid chromatography-mass spectrometry, LC-MS）检测方法，对干血斑（dried blood spot, DBS）样本中的两种生物素缺乏症关键生物标志物进行定量分析，以实现该病症的高效临床管理。
**材料与方法：** 本方法基于液相色谱-质谱系统开发，采用五氟苯基色谱柱，以甲醇与水的混合体系为流动相进行等度洗脱。参照相关行业指南对该方法开展了全面的方法学验证。
**结果与结论：** 本研究评估了干血斑检测法与血浆检测法的结果相关性，以明确两种方法的可互换性。所建立的方法已成功应用于印度南部沿海乌杜皮（Udupi）人群的上述两种生物标志物参考区间建立工作。
生物素缺乏可引发多种临床并发症，包括生长发育受阻、免疫功能受损、抑郁、肌痛，严重时可导致死亡。该病症可通过外源性补充生物素进行有效管控，早期检测对生物素缺乏症的临床管理至关重要。
本文详述了一种用于生物素缺乏症早期检测的综合性检测方案：该方法采用微创的干血斑采样方式，适配脆弱的新生儿群体。本液相色谱-质谱方法可同时定量检测干血斑中的3-羟基异戊酸（3-hydroxyisovaleric acid, 3-HIV）与3-羟基异戊酰肉碱（3-hydroxyisovaleryl carnitine, 3-HIC），可为新生儿及婴幼儿的生物素缺乏症诊断与预防提供全面的分析支撑。该方法具备简便易行、选择性优异、快速高效、特异性强且灵敏度极高的特点。
本研究对该方法的准确度、精密度、回收率、基质效应、血细胞比容效应、稳定性以及采血量/打孔位置效应均进行了系统性验证。结果显示，该方法线性关系良好（R²>0.99），灵敏度表现优异：3-HIV与3-HIC的定量下限（lower limit of quantitation, LLOQ）分别为0.088 μM与0.040 μM。两种分析物的质控（quality control, QC）样本回收率均处于可接受范围内。干血斑与血浆检测结果之间相关性较强，偏差极小。本方法已成功应用于印度南部乌杜皮人群的参考区间建立，该人群中3-HIV的参考区间为0.06–18.59 μM，3-HIC为0.01–0.49 μM。
本研究新开发的检测方法有望成为临床场景中生物素缺乏症确诊的可靠工具。