Eriocheir sinensis is a unique freshwater crab found in China, which is well known for its rich nutrition and sweet and delicious taste. Free amino acids in Eriocheir sinensis are not only important nutrients but also are closely related to their unique taste and aroma. Therefore, the determination of the free amino acid contents in Eriocheir sinensis is of great significance for product quality evaluation, flavor research, authenticity, and origin identification. Herein we proposed an ultra-high performance liquid chromatography-high resolution mass spectrometry (UHPLC-HRMS)-based method for the determination of 17 free amino acids in Eriocheir sinensis. First, 5 g of the Eriocheir sinensis sample was weighed into a 50-mL polypropylene centrifuge tube. Then, 10 mL of extraction solvents was added to the centrifuge tube, and the resultant solution was mixed well using a vortex mixer. We compared a variety of solvents and finally selected 5%(v/v) perchloric acid aqueous solution as the optimum extraction solvent. The supernatant was transferred to another polypropylene centrifuge tube after centrifuging at 8000 r/min for 5 min. The extraction procedure was repeated according to the above-mentioned steps, and the extraction solution was combined with the supernatant. The extracts were then adjusted to pH 6.5 with 1 mol/L potassium hydroxide solution, and were diluted to 50 mL with water. After filtering by both qualitative filter paper and a 0.45-μm polyether sulfone syringe filter, the extracts were determined by UHPLC-HRMS. We compared three types of mobile phases and chose 0.1%(v/v) formic acid aqueous solution mixed with acetonitrile as the optimum one. Precise parent ion and ion source parameters were also optimized. The 17 analytes, viz. aspartic acid, threonine, serine, glutamic acid, proline, cystine, valine, methionine, isoleucine, leucine, tyrosine, phenylalanine, lysine, arginine, glycine, alanine, and histidine, were separated on an XDB-C18 column (100 mm×4.6 mm, 1.7 μm) with gradient elution. The amino acids were then detected by HRMS in electrospray ionization and selected ion monitoring modes, and the analytes were quantified using external standards. The instrumental detection limit (IDL) and the instrumental quantification limit (IQL) were 0.3 mg/L and 1.0 mg/L, respectively. The linear correlation coefficients were all above 0.9990 in the concentration range of 10.0-200.0 mg/kg. Three levels of free amino acid standards were spiked into the edible parts of Eriocheir sinensis. The recoveries of the amino acids were between 78.4% and 105.3%. The intra-sample, intra-day, and inter-day repeatabilities were below 4.2%, 5.2%, and 11.4%, respectively, which were within reasonable ranges. Twenty samples of Eriocheir sinensis were tested using the proposed method. Thus, in this study, we developed an alternative method for the determination of free amino acids in Eriocheir sinensis with simple pretreatment, good selectivity, and high accuracy.
游离氨基酸不仅是一类重要的营养物质,更与中华绒螯蟹独特的滋味和香味密切相关。对游离氨基酸含量进行测定,能为中华绒螯蟹产品的品质评价和风味研究提供有价值的信息。该工作通过优选提取溶剂、优化仪器测定参数,最终使用5%(v/v)高氯酸水溶液提取目标物,采用XDB-C18色谱柱(100 mm×4.6 mm, 1.7 μm),以0.1%(v/v)甲酸水溶液-乙腈为流动相进行梯度洗脱,在电喷雾正离子电离、选择离子扫描模式下进行检测,外标法定量,建立了超高效液相色谱-高分辨质谱(UHPLC-HRMS)测定中华绒螯蟹中17种游离氨基酸含量的方法。结果表明,17种氨基酸在10.0~200.0 mg/L范围内线性关系良好,相关系数(R2)≥0.9990,仪器检出限为0.3 mg/L,仪器定量限为1.0 mg/L。在中华绒螯蟹可食部分中按三水平进行加标回收试验,各目标物的回收率在78.4%~105.3%之间。通过对仪器和方法精密度进行评估,发现17种氨基酸测定仪器重复性的RSD≤4.2%,方法重复性试验的RSD≤5.2%,再现性试验的RSD≤11.4%,精密度数据均在合理范围内。利用该研究建立的检测方法对20个中华绒螯蟹样本进行了实际样品检测,总结了17种游离氨基酸在蟹肉、蟹膏、蟹黄中的分布特点,验证了该方法是一种前处理简便、选择性好、准确度高的检测技术。
Keywords: Eriocheir sinensis; free amino acid; ultra-high performance liquid chromatography-high resolution mass spectrometry (UHPLC-HRMS).