1,2-Diacyl-sn-glycero-3-phospho-N-acyl-ethanolamines (NAPE) are low abundance phospholipids but important constituents of intracellular membranes of plant tissues, responsible for generating bioactive N-acylethanolamine (NAE), which participates in several physiological processes such as regulation of seed germination and protection against pathogenic attacks. From an analytical point of view, the critical aspect of these bioactive lipids lies in the determination of fatty acyl chains located in sn-1/sn-2 position on the glycerol backbone (O-linked), along with the amide-bound (N-linked) fatty acyl chain. Here, the identity and occurrence of NAPE in lipid extracts of lupin seeds (Lupinus luteus L.) was assessed by electrospray ionization in negative ion mode upon reversed-phase liquid chromatography (RPLC-ESI) coupled to mass spectrometry (MS) either at high- (i.e., Orbitrap FTMS) or low- (linear ion trap, LIT) resolution/accuracy. Collisional induced dissociation (CID)-tandem MS and MS3 acquisitions of chemically prepared NAPE allowed to unequivocally recognize the N-linked fatty acyl chain and to establish the diagnostic product ions that were successfully applied to identify NAPE in lipid extracts of yellow lupin seeds. The most abundant NAPE species were those containing N-acyl groups C18:1, C18:2; a minor prevalence was found for C16:0, C18:0, and C18:3, and almost the same acyl chains O-linked on the glycerol backbone in several sn-1/sn-2 combinations were observed. The positional isomers of NAPE species were identified as deprotonated molecules ([M-H]-) at m/z 978.7541 (three isomers 52:3), m/z 980.7694 (two isomers 52:2), m/z 1002.7535 (four isomers 54:5), m/z 1004.7686 (two isomers 54:4), m/z 1006.7837 (two isomers 54:3), and m/z 1008.8026 (single isomer 54:2). The total amount of NAPE in lupin seeds ranged in the interval of 2.00 ± 0.13 mg/g dw, in agreement with other edible legumes. We anticipate our approach to be a robust assessment method potentially applicable to biological extracts containing NAPE species and can provide comprehensive profiles and contents.

中文翻译：

---

N-酰基磷脂酰乙醇胺 (NAPE) 通过液相色谱/电喷雾电离与多级质谱的区域化学归属及其在羽扇豆种子提取物中的应用。

1,2-Diacyl-sn-glycero-3-phospho-N-acyl-ethanolamines (NAPE) 是低丰度的磷脂，是植物组织细胞内膜的重要成分，负责生成具有生物活性的 N-酰基乙醇胺 (NAE)，参与几种生理过程，例如调节种子萌发和防止病原体攻击。从分析的角度来看，这些生物活性脂质的关键方面在于确定位于甘油主链上 sn-1/sn-2 位置的脂肪酰基链（O 连接），以及酰胺结合的（N -连接的）脂肪酰基链。在这里，羽扇豆种子 (Lupinus luteus L. ) 通过反相液相色谱 (RPLC-ESI) 与质谱 (MS) 以高（即 Orbitrap FTMS）或低（线性离子阱，LIT）分辨率耦合的负离子模式电喷雾电离进行评估/准确性。化学制备的 NAPE 的碰撞诱导解离 (CID)-串联 MS 和 MS3 采集允许明确识别 N 连接的脂肪酰基链并建立诊断产物离子，该离子成功应用于识别黄色羽扇豆种子脂质提取物中的 NAPE。最丰富的 NAPE 种类是那些含有 N-酰基 C18:1、C18:2 的种类；发现 C16:0、C18:0 和 C18:3 的流行率较低，并且在几个 sn-1/sn-2 组合中观察到几乎相同的酰基链 O-连接在甘油主链上。NAPE 物质的位置异构体被鉴定为去质子化分子 ([MH]-)，m/z 978.7541（三种异构体 52:3）、m/z 980.7694（两种异构体 52:2）、m/z 1002.7535（四种异构体 54 :5)、m/z 1004.7686（两种异构体 54:4）、m/z 1006.7837（两种异构体 54:3）和 m/z 1008.8026（单一异构体 54:2）。羽扇豆种子中 NAPE 的总量在 2.00 ± 0.13 mg/g dw 的区间内，与其他可食用豆类一致。我们预计我们的方法是一种强大的评估方法，可能适用于含有 NAPE 物种的生物提取物，并且可以提供全面的概况和内容。羽扇豆种子中 NAPE 的总量在 2.00 ± 0.13 mg/g dw 的区间内，与其他可食用豆类一致。我们预计我们的方法是一种强大的评估方法，可能适用于含有 NAPE 物种的生物提取物，并且可以提供全面的概况和内容。羽扇豆种子中 NAPE 的总量在 2.00 ± 0.13 mg/g dw 的区间内，与其他可食用豆类一致。我们预计我们的方法是一种强大的评估方法，可能适用于含有 NAPE 物种的生物提取物，并且可以提供全面的概况和内容。