Castro Juliana de L, Martucci Maria Elvira P, Pereira Henrique M G, de Sousa Valéria P
Department of Drugs and Medicines, Faculty of Pharmacy, Federal University of Rio de Janeiro, Av. Carlos Chagas Filho, 373, CCS, Cidade Universitária, Rio de Janeiro, 21941-902, Brazil.
Institute of Chemistry, LBCD-LADETEC, Federal University of Rio de Janeiro, Av. Horácio Macedo, 1280, Pólo de Química, Bloco C, Cidade Universitária, Rio de Janeiro, 21941-598, Brazil.
J Mass Spectrom. 2020 Oct;55(10):e4593. doi: 10.1002/jms.4593.
Dermorphin is a peptide with analgesic actions similar to morphine, but with greater effect and less potential to cause tolerance. The use of dermorphin has been documented in race horses, and its use in humans has already been reported. Considering the potential advantages from the use of dermorphin over morphine, a method to monitor it, and its main metabolite dermorphin (1-4) in humans becomes necessary for doping control. Here, we present two orthogonal methods for this purpose: a high-throughput liquid chromatography coupled to high-resolution mass spectrometry (HRMS) as an initial testing procedure and liquid chromatography-tandem mass spectrometry (MS/MS) in the selected reaction monitoring (SRM) acquisition mode for a confirmation procedure. For urine samples, pretreatment through a mixed-mode weak cation-exchange solid-phase extraction emerged as an effective approach to extract peptides from the biological sample. For the HRMS analysis, a full-MS scan acquisition mode was selected to detect the exact masses of dermorphin and dermorphin (1-4) at m/z 803.37226 and 457.20816, respectively. The SRM method used in the MS/MS confirmation protocol presented high specificity and sensitivity. The selected product ions for dermorphin were 602.2, 202.1 and 574.3 and for dermorphin (1-4) were 207.1, 223.1, and 235.1. Both methods were evaluated for specificity, repeatability, carryover, matrix effects, and recovery. No carryover and matrix effects were detected. The limit of detection for initial testing procedure and the limit of identification for confirmation procedure was 2.5 ng/ml. Also, specificity and robustness were acceptable for the application. Together, the developed methods proved to be efficient for the analysis of dermorphin and metabolite for human doping control purpose.
强啡肽是一种具有与吗啡相似镇痛作用的肽，但效果更强且产生耐受性的可能性更小。强啡肽在赛马中的使用已有记录，其在人类中的使用也已被报道。考虑到与吗啡相比，使用强啡肽可能具有的优势，有必要开发一种监测人体中强啡肽及其主要代谢物强啡肽（1 - 4）的方法，以进行兴奋剂检测。在此，我们为此目的提出两种正交方法：一种是高通量液相色谱与高分辨率质谱联用（HRMS）作为初始检测程序，另一种是在选择反应监测（SRM）采集模式下的液相色谱 - 串联质谱（MS/MS）作为确证程序。对于尿液样本，通过混合模式弱阳离子交换固相萃取进行预处理是从生物样本中提取肽的有效方法。对于HRMS分析，选择全扫描采集模式分别检测强啡肽和强啡肽（1 - 4）的精确质量，其质荷比分别为803.37226和457.20816。MS/MS确证方案中使用的SRM方法具有高特异性和高灵敏度。强啡肽选择的产物离子为602.2、202.1和574.3，强啡肽（1 - 4）选择的产物离子为207.1、223.1和235.1。对这两种方法的特异性、重复性、残留、基质效应和回收率进行了评估。未检测到残留和基质效应。初始检测程序的检测限和确证程序的识别限均为2.5 ng/ml。此外，该方法的特异性和稳健性适用于实际应用。总之，所开发的方法被证明对于分析人体兴奋剂检测中的强啡肽及其代谢物是有效的。
