Institute of Forensic Medicine, Jena University Hospital, Jena, Germany.
Institute of Forensic Medicine, Jena University Hospital, Jena, Germany.
J Chromatogr A. 2018 Jul 27;1560:35-44. doi: 10.1016/j.chroma.2018.05.013. Epub 2018 May 7.
Endophyte fungi (e.g. Epichloë ssp. and Neotyphodium ssp.) in symbiosis with pasture grasses (e.g. Festuca arundinacaea and Lolium perenne) can produce toxic alkaloids, which are suspected to be involved in equine diseases such as fescue toxicosis, ryegrass staggers, and equine fescue oedema. The aim of this study was, therefore, to develop and validate a quantification method for these and related alkaloids: ergocristine, ergocryptine, ergotamine, ergovaline, lolitrem B, lysergic acid, N-acetylloline, N-formylloline, peramine, and paxilline in horse serum. Horse serum samples (1.5mL) were worked up by solid-phase extraction (OASIS HLB). The extracts were analyzed by ultra high performance liquid chromatography-high resolution tandem mass spectrometry (UHPLC-HRMS/MS). Chromatographic separation was achieved by gradient elution with ammonium formate buffer and acetonitrile on a RP18 column (100×2.1mm; 1.7μm). HRMS/MS detection was performed on a QExactive Focus instrument with heated positive electrospray ionization and operated in the parallel reaction monitoring (PRM) mode. Method validation included evaluation of selectivity, matrix effect, recovery, linearity, limit of quantification (LOQ), limit of detection (LOD), accuracy, and stability. With exception of lolitrem B solid phase extraction yielded high recoveries (73.6-104.6%) for all analytes. Chromatographic separation of all analytes was achieved with a run time of 25min. HRMS/MS allowed sensitive detection with LODs ranging from 0.05 to 0.5ng/mL and LOQs from 0.1 to 1.0ng/mL. Selectivity experiments showed no interferences from matrix or IS, but N-acetylloline and N-formylloline were found to be ubiquitous in horse serum. Newborn calf serum was therefore used as surrogate matrix for the validation study. Calibration ranges were analyte-dependent and in total covered concentrations from 0.1 to 50ng/mL. Lolitrem B and paxilline could be sensitively detected, but did not meet quantification requirements. For the other analytes, accuracy and precision were shown for 3 different concentrations (QC low, medium, high) with acceptable bias (-10, 5%-7.9%) and precision (CV 2.6%-12.5%). Matrix effects varied from 55.0% to 121% (RSD 7.8-18.5%) and were adequately compensated by IS. Matrix effects of N-acetylloline and N-formylloline could only be estimated in newborn calf serum (n=1) and ranged from 52.5-88.3%. All analytes were stable under autosampler conditions and over 3 freeze and thaw cycles. Applicability was proven by analyzing authentic horse serum samples (n=24). In conclusion, the presented method allows a sensitive detection of ergocrisitine, ergocryptine, ergotamine, ergovaline, lolitrem B, lysergic acid, N-acetylloline, N-formylloline, peramine, and paxilline in horse serum and reliable quantification of all but lolitrem B and paxilline.
内生真菌（如 Epichloë 属和 Neotyphodium 属）与牧草（如高羊茅和黑麦草）共生可产生有毒生物碱，这些生物碱被怀疑与马的疾病有关，如雀麦中毒、黑麦草中毒和马的雀麦水肿。因此，本研究的目的是开发和验证一种定量分析这些和相关生物碱的方法：马血清中的麦角克碱、麦角隐亭、麦角新碱、麦角卡宁、麦角灵 B、麦角酸、N-乙酰洛林、N-甲酰洛林、佩兰素和派利灵。马血清样品（1.5mL）通过固相萃取（OASIS HLB）进行处理。提取物通过梯度洗脱用甲酸铵缓冲液和乙腈在 RP18 柱（100×2.1mm；1.7μm）上进行分析。HRMS/MS 检测在 QExactive Focus 仪器上进行，采用加热正电喷雾电离，在平行反应监测（PRM）模式下运行。方法验证包括选择性、基质效应、回收率、线性、定量限（LOQ）、检测限（LOD）、准确性和稳定性的评价。除了麦角灵 B 外，固相萃取对所有分析物的回收率（73.6-104.6%）都很高。所有分析物的色谱分离在 25 分钟内完成。HRMS/MS 允许以 0.05 至 0.5ng/mL 的 LOD 和 0.1 至 1.0ng/mL 的 LOQ 进行灵敏检测。选择性实验表明基质或 IS 没有干扰，但 N-乙酰洛林和 N-甲酰洛林在马血清中普遍存在。因此，新生小牛血清被用作验证研究的替代基质。校准范围取决于分析物，总浓度范围为 0.1 至 50ng/mL。可以灵敏地检测到麦角灵 B 和派利灵，但不符合定量要求。对于其他分析物，在可接受的偏差（-10、5%-7.9%）和精密度（CV 2.6%-12.5%）下，对 3 个不同浓度（QC 低、中、高）进行了准确性和精密度的研究。基质效应在 55.0%至 121%之间（RSD 7.8-18.5%），并通过 IS 得到充分补偿。N-乙酰洛林和 N-甲酰洛林的基质效应只能在新生小牛血清（n=1）中进行估计，范围在 52.5-88.3%之间。所有分析物在自动进样器条件下和 3 次冻融循环中均稳定。通过分析真实的马血清样本（n=24）证明了该方法的适用性。总之，该方法能够灵敏地检测马血清中的麦角克碱、麦角隐亭、麦角新碱、麦角卡宁、麦角灵 B、麦角酸、N-乙酰洛林、N-甲酰洛林、佩兰素和派利灵，并可靠地定量除麦角灵 B 和派利灵以外的所有分析物。
