干血斑(DBS)技术是一种简单方便的采集方法,运输,把血样储存在滤纸上,在临床上有许多应用,研究,和公共卫生环境。这项技术在法医学领域越来越受欢迎,因为它有助于快速分析血液样本中的违禁药物,并在酒后驾车筛查等毒理学场景中提供显著优势,药物滥用检测,和兴奋剂检测。然而,缺乏规范的制度,其稳定性和可靠性尚未得到深入研究和论证,限制了其在我国司法实践中的应用。可以制备DBS样品,存储,并以各种方式进行了分析,所有这些都可能显著影响结果。在这项研究中,我们开发了一种基于超高效液相色谱-串联质谱(UHPLC-MS/MS)的方法,预处理,分析,和存储DBS样品。进行了彻底的调查,以检查最佳的制备条件,包括血斑基质,干燥技术,和预处理参数,如溶剂和提取方法。此外,分析条件,如流动相系统和洗脱梯度,是为了促进甲基苯丙胺的定量检测而建立的,利多卡因,氯胺酮,芬太尼,DBS和全血样本中的地西泮。储存条件的影响,比如温度,湿度,湿度和密封,还检查了DBS和全血样品的分析结果。结果表明,利多卡因和芬太尼在0.5-100ng/mL范围内具有很强的线性关系。同样,甲基苯丙胺,氯胺酮,和地西泮在2-100ng/mL范围内表现出良好的线性。测定系数(r2)范围为0.9983~0.9997,检测限范围为0.2~0.5ng/mL,表明高度的相关性和敏感性。稳定性测试表明,五种目标物质在DBS中保持稳定60天,测得的含量与标称值偏离15%。此外,DBS样本的测量结果与全血样本的测量结果高度相似,平均百分比差异为4.44%,3.50%,7.66%,5.10%,芬太尼为5.25%,地西泮,氯胺酮,利多卡因,和甲基苯丙胺,分别。在整个60天的储存期间,维持温度-20和4℃,以及密封和干燥储存,没有必要。室温是DBS样品最实用的储存环境。每个目标的结果显示全血和DBS样品之间的浓度差异非常小,表明DBS样品适用于血液中的药物和毒物分析。此外,DBS与全血样本表现出高度的定量一致性,使它们适合保存血液样品的基质。由于DBS样品易于处理和存储,实现了血液样本的轻量化保存,为公安实践中血液样本的分析和保存提供了一种新的解决方案。我们建议在使用DBS进行分析之前进行全面验证,特别是在量化方面,确保司法结果的可靠性。
Dried blood spot (DBS) technology is a simple and convenient method for collecting, transporting, and storing blood samples on filter paper, and has numerous applications in the clinical, research, and public health settings. This technique is gaining popularity in the field of forensic science because it facilitates the rapid analysis of prohibited drugs in blood samples and offers significant advantages in toxicology scenarios such as drinking-driving screening, drug abuse detection, and doping detection. However, the lack of a standardized system and the fact that its stability and reliability have not been thoroughly researched and demonstrated limit its application in judicial practice in China. DBS samples can be prepared, stored, and analyzed in various ways, all of which may significantly affect the results. In this study, we developed a method based on ultra-high performance liquid chromatography-tandem mass spectrometry (UHPLC-MS/MS) that focuses on the preparation, pretreatment, analysis, and storage of DBS samples. A thorough investigation was conducted to examine the optimal preparation conditions, including the blood spot matrix, drying technique, and preprocessing parameters, such as the solvent and extraction method. Moreover, the analytical conditions, such as the mobile phase system and elution gradient, were established to facilitate the quantitative detection of methamphetamine, lidocaine, ketamine, fentanyl, and
diazepam in both DBS and whole-blood samples. The impact of storage conditions, such as the temperature, humidity, and sealing, on the analytical results of the DBS and whole-blood samples was also examined. The results showed a strong linear relationship for lidocaine and fentanyl within the range of 0.5-100 ng/mL. Similarly, methamphetamine, ketamine, and
diazepam exhibited good linearity within the range of 2-100 ng/mL. The coefficients of determination (r2) ranged from 0.9983 to 0.9997, and the limits of detection ranged from 0.2 to 0.5 ng/mL, indicating a high degree of correlation and sensitivity. Stability tests demonstrated that the five target substances remained stable in the DBS for 60 days, with the measured contents deviating from the nominal values by 15%. Moreover, the measurement results of the DBS samples were highly similar to those of the whole-blood samples, with mean percentage differences of 4.44%, 3.50%, 7.66%, 5.10%, and 5.25% for fentanyl,
diazepam, ketamine, lidocaine, and methamphetamine, respectively. Throughout the 60-day storage period, the maintenance of temperatures of -20 and 4 ℃, as well as sealing and dry storage, was not necessary. Room temperature was the most practical storage environment for the DBS samples. The results for each target showed very small concentration differences between the whole-blood and DBS samples, indicating that the DBS samples were suitable for drug and poison analysis in blood. Furthermore, the DBSs exhibited high quantitative consistency with the whole-blood samples, rendering them suitable matrices for preserving blood samples. Because DBS samples are easy to handle and store, they can realize the lightweight preservation of blood samples and provide a novel solution for the analysis and preservation of blood samples in public security practice. We recommend conducting comprehensive validations before utilizing DBS for analysis, particularly in terms of quantification, to ensure the judicial reliability of the results.