Department of Pathology, Center for Advanced Laboratory Medicine, University of California, San Diego Health Systems, San Diego, CA;
Waters Corp., Pleasanton, CA.
Clin Chem. 2014 Aug;60(8):1115-25. doi: 10.1373/clinchem.2014.222976. Epub 2014 Jun 10.
High-resolution mass spectrometry (HRMS) has the potential to supplement other drug screening platforms used in toxicology laboratories. HRMS offers high analytical specificity, which can be further enhanced by incorporating a fragment ion for each analyte. The ability to obtain precursor ions and fragment ions using elevated collision energies (MS(E)) can help improve the specificity of HRMS methods.
We developed a broad-spectrum screening method on an ultraperformance liquid chromatography TOF mass spectrometer (UPLC-TOF-MS) using the MS(E) mode. A diverse set of patient samples were subjected to a simple dilute, hydrolyze, and shoot protocol and analyzed in a blind manner. Data were processed with 3 sets of criteria with increasing stringency, and the results were compared with the reference laboratory results.
A combination of retention time match (±0.2 min), a protonated analyte, and fragment ion mass accuracy of ±5 ppm produced zero false-positive results. Using these criteria, we confirmed 92% (253/275) of true positives. The positive confirmation rate increased to 98% (270/275) when the requirement for a fragment ion was dropped, but also produced 53 false positives. A total of 136 additional positive drug findings not identified by the reference methods were identified with the UPLC-TOF-MS.
MS(E) provides a unique way to incorporate fragment ion information without the need of precursor ion selection. A primary limitation of requiring a fragment ion for positive identification was that certain drug classes required high-energy collisions, which formed many fragment ions of low abundance that were not readily detected.
高分辨率质谱（HRMS）有可能补充毒理学实验室中使用的其他药物筛选平台。HRMS 具有高分析特异性，通过为每个分析物引入碎片离子，可以进一步增强其特异性。使用升高的碰撞能（MS(E)）获得前体离子和碎片离子的能力可以帮助提高 HRMS 方法的特异性。
我们在超高效液相色谱飞行时间质谱仪（UPLC-TOF-MS）上使用 MS(E)模式开发了一种广谱筛选方法。对一组不同的患者样本进行简单的稀释、水解和拍摄方案处理，并进行盲法分析。使用 3 组具有不同严格程度的标准进行数据处理，并将结果与参考实验室结果进行比较。
保留时间匹配（±0.2 分钟）、质子化分析物和碎片离子质量精度为±5ppm 的组合产生了零个假阳性结果。使用这些标准，我们确认了 92%（253/275）的真阳性。当降低对碎片离子的要求时，阳性确认率增加到 98%（270/275），但也产生了 53 个假阳性。总共鉴定出 136 种额外的、未被参考方法识别的阳性药物发现。
MS(E)提供了一种独特的方法来结合碎片离子信息，而无需选择前体离子。需要碎片离子进行阳性鉴定的一个主要限制是某些药物类别需要高能量碰撞，这会形成许多低丰度的碎片离子，不易检测。
