Center for Preventive Doping Research and Institute of Biochemistry, German Sport University Cologne, Am Sportpark Müngersdorf 6, 50933, Cologne, Germany.
Drug Test Anal. 2014 Nov-Dec;6(11-12):1125-32. doi: 10.1002/dta.1710. Epub 2014 Sep 14.
The qualitative and quantitative determination of insulin from human blood samples is an emerging topic in doping controls as well as in other related disciplines (e.g. forensics). Beside the therapeutic use, insulin represents a prohibited, performance enhancing substance in sports drug testing. In both cases accurate, sensitive, specific, and unambiguous determination of the target peptide is of the utmost importance. The challenges concerning identifying insulins in blood by liquid chromatography coupled to ion mobility mass spectrometry (LC-IM-MS) are detecting the basal concentrations of approximately 0.2 ng/mL and covering the hyperinsulinaemic clamps at > 3 ng/mL simultaneously using up to 200 μL of plasma or serum. This is achieved by immunoaffinity purification of the insulins with magnetic beads and subsequent separation by micro-scale liquid chromatography coupled to ion mobility / high resolution mass spectrometry. The method includes human insulin as well as the synthetic or animal analogues insulin aspart, glulisine, glargine, detemir, lispro, bovine, and porcine insulin. The method validation shows reliable results considering specificity, limit of detection (0.2 ng/mL except for detemir: 0.8 ng/mL), limit of quantification (0.5 ng/mL for human insulin), precision (CV < 20%), linearity (r > 0.99), recovery, accuracy (>90%), robustness (plasma/serum), and ion suppression. For quantification of human insulin a labelled internal standard ([[(2) H10 ]-Leu(B6,B11,B15,B17) ] - human Insulin) is introduced. By means of the additional ion mobility separation of the different analogues, the chromatographic run time is shortened to 8 min without losing specificity. As proof-of-concept, the procedure was successfully applied to different blood specimens from diabetic patients receiving recombinant synthetic analogues.
从人血样本中定性和定量测定胰岛素是兴奋剂检测以及其他相关学科（例如法医学）中的一个新兴课题。除了治疗用途外，胰岛素在运动药物检测中也是一种被禁止的、提高运动表现的物质。在这两种情况下，准确、灵敏、特异和明确地测定目标肽都是至关重要的。通过液相色谱-离子淌度质谱联用（LC-IM-MS）鉴定血液中的胰岛素面临的挑战是，使用多达 200μL 的血浆或血清同时检测大约 0.2ng/mL 的基础浓度和覆盖 3ng/mL 以上的高胰岛素钳夹。这是通过用磁性珠进行胰岛素的免疫亲和纯化，然后通过微尺度液相色谱-离子淌度/高分辨率质谱联用进行分离来实现的。该方法包括人胰岛素以及合成或动物类似物胰岛素天冬氨酸、赖脯胰岛素、甘精胰岛素、地特胰岛素、门冬胰岛素、牛胰岛素和猪胰岛素。方法验证表明，该方法具有可靠的结果，考虑到特异性、检测限（除地特胰岛素外为 0.2ng/mL：0.8ng/mL）、定量限（人胰岛素为 0.5ng/mL）、精密度（CV<20%）、线性（r>0.99）、回收率、准确性（>90%）、稳健性（血浆/血清）和离子抑制。为了定量测定人胰岛素，引入了一种标记的内标（[[（2）H10]-Leu（B6，B11，B15，B17）]-人胰岛素）。通过不同类似物的额外离子淌度分离，可以将色谱运行时间缩短至 8 分钟，而不会失去特异性。作为概念验证，该程序成功应用于接受重组合成类似物的糖尿病患者的不同血液样本。
