Supplementary information files for Development of a rapid, in-situ analysis method using sheath-flow probe electrospray ionisation-mass spectrometry for the direct identification of cocaine metabolites in dried blood spots

Supplementary files for article Development of a rapid, in-situ analysis method using sheath-flow probe electrospray ionisation-mass spectrometry for the direct identification of cocaine metabolites in dried blood spots

Rationale
Small amounts of biofluid samples are frequently found at crime scenes; however, existing gold standard methods such as LC–MS frequently require destructive extraction of the sample before a time-consuming analysis which puts strain on forensic analysis providers and can preclude further sample analysis. This study presents the application of sheath-flow probe electrospray ionization-mass spectrometry (sfPESI–MS) to the direct analysis of drug metabolites in dried blood spots (DBS) as a high throughput, minimally destructive alternative.

Methods
A rapid direct analysis method using a sfPESI ionisation source coupled to an Orbitrap Exactive mass spectrometer was applied to detect cocaine metabolites (benzoylecgonine, BZE, cocaethylene, CE, and ecgonine methyl ester, EME) from DBS. An optimisation study exploring the use of different chemical modifiers (formic acid and sodium acetate) in the sfPESI probe extraction solvent was conducted to enhance the sensitivity and reproducibility of the sfPESI–MS method.

Results
Optimisation of the extraction solvent significantly enhanced the sensitivity and reproducibility of the sfPESI–MS method. A quantitative response over a five-point calibration range 0.5 to 10 μg/ml was obtained for BZE (R2 = 0.9979) and CE (R2 = 0.9948). Limits of detection (LOD) of 1.31, 0.29 and 0.15 μg/ml were achieved for EME, BZE and CE, respectively, from 48 h aged DBSs with % RSD (relative standard deviation) across the calibration range ranging between 19%–28% for [BZE + H]+, 13%–21% for [CE + H]+ and 12%–29% for [EME + H]+.

Conclusions
A rapid (< 20 s) quantitative method for the direct analysis of cocaine metabolites from DBS which requires no prior sample preparation was developed. Although the LOD achieved for BZE (LOD: 0.29 μg/ml) was above the UK threshold limit of exposure for drug driving (0.05 μg/ml), the method may be suitable for use in identifying overdose in forensic analysis.

关于使用鞘流探针电喷雾电离-质谱法快速原位分析干血斑中可卡因代谢物的补充文件

研究缘起
在犯罪现场，生物流体样本的微量存在是常见的；然而，现有的金标准方法如液相色谱-质谱法，通常需要在对样品进行耗时分析之前进行破坏性提取，这给法医分析服务提供商带来了压力，并可能阻止进一步的样本分析。本研究展示了鞘流探针电喷雾电离-质谱法（sfPESI-MS）在干血斑（DBS）中直接分析药物代谢物（苯甲酰伪麻黄碱，BZE，可卡因乙酯，CE，以及伪麻黄碱甲酯，EME）的应用，作为一种高吞吐量、最小破坏性的替代方案。

研究方法
将sfPESI电离源与Orbitrap Exactive质谱仪相连的快速直接分析方法应用于从DBS中检测可卡因代谢物。进行了一项优化研究，探讨在sfPESI探针提取溶剂中不同化学修饰剂（甲酸和醋酸钠）的使用，以提高sfPESI-MS方法的灵敏度和重现性。

研究结果
提取溶剂的优化显著提高了sfPESI-MS方法的灵敏度和重现性。在BZE（R²=0.9979）和CE（R²=0.9948）的五个点校准范围内获得了定量响应，范围为0.5至10 μg/ml。对于EME、BZE和CE，分别从48小时老化的DBS中达到了检测限（LOD）为1.31、0.29和0.15 μg/ml，校准范围内的相对标准偏差（% RSD）分别为[BZE+H]+的19%–28%，[CE+H]+的13%–21%，以及[EME+H]+的12%–29%。

研究结论
开发了一种快速（< 20秒）的定量方法，用于直接分析干血斑中的可卡因代谢物，无需预先准备样品。尽管BZE的检测限（LOD：0.29 μg/ml）高于英国药物驾驶的暴露阈值（0.05 μg/ml），但该方法可能适用于法医分析中识别药物过量。