Endocannabinoid Research Group, Reproductive Science Section, Department of Cancer Studies and Molecular Medicine, University of Leicester, Leicester LE2 7LX, United Kingdom.
J Chromatogr B Analyt Technol Biomed Life Sci. 2010 Dec 1;878(31):3231-7. doi: 10.1016/j.jchromb.2010.09.024. Epub 2010 Oct 1.
The endocannabinoids anandamide, palmitoylethanolamide and oleoylethanolamide have been detected in human seminal plasma and are bioactive lipids implicated in regulation of sperm motility, capacitation and acrosome reaction. Several methods exist for endocannabinoid quantification but none have been validated for measurement in human seminal plasma. We describe sensitive, robust, reproducible solid phase and isotope-dilution UHPLC-ESI-MS/MS methods for the extraction and quantification of anandamide, palmitoylethanolamide and oleoylethanolamide in human seminal plasma. Precision and accuracy were evaluated using pooled seminal plasma over a 4 day period. For all analytes, the inter- and intraday precision (CV%) was between 6.6-17.7% and 6.3-12.5%, respectively. Analyses were linear over the range 0.237-19nM for anandamide and oleoylethanolamide and 0.9-76nM for PEA. Limits of detection (signal-to-noise >3) were 50, 100 and 100fmol/mL and limits of quantification (signal-to-noise >10) were 100, 200 and 200fmol/mL, respectively for anandamide, palmitoylethanolamide and oleoylethanolamide. Anandamide and oleoylethanolamide were stable at -80°C for up to 4 weeks, but palmitoylethanolamide declined significantly. We assessed seminal plasma from 40 human donors with normozoospermia and found mean (inter-quartile range) concentrations of 0.21nM (0.09-0.27), 1.785nM (0.48-2.32) and 15.54nM (7.05-16.31) for anandamide, oleoylethanolamide and palmitoylethanolamide, respectively. Consequently, this UHPLC-ESI-MS/MS method represents a rapid, reliable and reproducible technique for the analysis of these endocannabinoids in fresh seminal plasma.
内源性大麻素大麻酰胺、棕榈酰乙醇酰胺和油酰乙醇酰胺已在人精液中被检测到，它们是参与调节精子运动能力、获能和顶体反应的生物活性脂质。有几种方法可以用于内源性大麻素的定量，但没有一种方法已经过验证可用于测量人精液中的内源性大麻素。我们描述了一种灵敏、稳健、重现性好的固相和同位素稀释 UHPLC-ESI-MS/MS 方法，用于提取和定量人精液中的大麻酰胺、棕榈酰乙醇酰胺和油酰乙醇酰胺。使用在 4 天内收集的混合精液评估精密度和准确度。对于所有分析物，日内和日间精密度（CV%）分别在 6.6-17.7%和 6.3-12.5%之间。分析物的线性范围分别为大麻酰胺和油酰乙醇酰胺的 0.237-19nM 和棕榈酰乙醇酰胺的 0.9-76nM。检测限（信噪比>3）分别为 50、100 和 100fmol/mL，定量限（信噪比>10）分别为 100、200 和 200fmol/mL，用于大麻酰胺、棕榈酰乙醇酰胺和油酰乙醇酰胺。大麻酰胺和油酰乙醇酰胺在-80°C 下可稳定 4 周，但棕榈酰乙醇酰胺显著下降。我们评估了 40 名具有正常精子计数的人类供体的精液样本，发现大麻酰胺、油酰乙醇酰胺和棕榈酰乙醇酰胺的平均（四分位距）浓度分别为 0.21nM（0.09-0.27）、1.785nM（0.48-2.32）和 15.54nM（7.05-16.31）。因此，这种 UHPLC-ESI-MS/MS 方法代表了一种快速、可靠和可重现的技术，用于分析新鲜精液中的这些内源性大麻素。
