以 2,3-萘二甲醛为荧光衍生试剂的高效液相色谱荧光检测分析尿液氨基酸,Separations

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以 2,3-萘二甲醛为荧光衍生试剂的高效液相色谱荧光检测分析尿液氨基酸
Separations ( IF 1.9 ) Pub Date : 2023-03-24 , DOI: 10.3390/separations10040224
Yuma Ishida ₁ , Ryoto Inoue ₁ , Makoto Tsunoda ₁

Affiliation

氨基酸参与体内各种化学反应，尿液中几种氨基酸的变化与某些疾病状态有关。因此，开发一种有效的尿液氨基酸分析方法有助于疾病的及时诊断。在这项研究中，我们开发了一种高效液相色谱 (HPLC) 荧光方法，使用荧光衍生化试剂 2,3-萘二甲醛 (NDA) 定量分析尿液氨基酸。选择 NDA 是因为它不需要加热进行反应，并且可以在短时间内发生反应，使其在临床环境中的使用变得可行。对反应温度、反应时间等衍生化条件进行了优化，发现反应在25℃下5 min即可完成。在梯度条件下，在十八烷基甲硅烷基 (ODS) 柱上研究了 NDA-氨基酸的分离。流动相是水-乙腈-三氟乙酸的混合物。十八个NDA-氨基酸（组氨酸（His）、精氨酸（Arg）、天冬酰胺（Asn）、谷氨酰胺（Gln）、瓜氨酸（Cit）、丝氨酸（Ser）、天冬氨酸（Asp）、苏氨酸（Thr）、谷氨酸（ Glu)、甘氨酸 (Gly)、酪氨酸 (Tyr)、丙氨酸 (Ala)、色氨酸 (Trp)、缬氨酸 (Val)、苯丙氨酸 (Phe)、异亮氨酸 (Ile)、亮氨酸 (Leu) 和 5-氨基戊酸（内部standard)) 在最佳条件下在 100 分钟内分离。校准曲线在每次进样 0.25–25 pmol 范围内显示出良好的线性，相关系数 > 0.998。NDA-氨基酸的定量限为 16.7-74.7 fmol。将开发的分析方法应用于人尿液样品，分析了 16 种氨基酸（His、Arg、Asn、Gln、Cit、Ser、Thr、Glu、Gly、Tyr、Ala、Trp、Val、Phe、Ile 和 Leu）量化。尿氨基酸浓度为 5–960 μM。使用这种方法进行的尿液氨基酸分析有望在临床上用作影响膀胱、泌尿道和肾脏的疾病的新型生物标志物。

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Analysis of Urinary Amino Acids by High-Performance Liquid Chromatography with Fluorescence Detection Using 2,3-Naphthalenedicarboxaldehyde as Fluorescence Derivatization Reagent

Amino acids are involved in various chemical reactions in vivo, and changes in several amino acids in urine are related to certain disease states. Therefore, developing an efficient method to analyze the amino acids in urine is useful in the timely diagnosis of diseases. In this study, we developed a high-performance liquid chromatography (HPLC) fluorescence method for the quantitative analysis of urinary amino acids using the fluorescence derivatization reagent 2,3-naphthalenedicarboxaldehyde (NDA). NDA was selected because it does not require heating for the reaction and can react within a short time, rendering its use in clinical settings feasible. The reaction temperature, reaction time, and other derivatization conditions were optimized, and the reaction was found to be completed in 5 min at 25 °C. The separation of NDA–amino acids was investigated on an octadecylsilyl (ODS) column under gradient conditions. The mobile phase was a mixture of water–acetonitrile–trifluoroacetic acid. Eighteen NDA–amino acids (histidine (His), arginine (Arg), asparagine (Asn), glutamine (Gln), citrulline (Cit), serine (Ser), aspartic acid (Asp), threonine (Thr), glutamic acid (Glu), glycine (Gly), tyrosine (Tyr), alanine (Ala), tryptophan (Trp), valine (Val), phenylalanine (Phe), isoleucine (Ile), leucine (Leu), and 5-aminovaleric acid (internal standard)) were separated within 100 min under optimal conditions. The calibration curves showed good linearity in the range of 0.25–25 pmol per injection with correlation coefficients of >0.998. The limits of quantification for NDA–amino acids were 16.7–74.7 fmol. The developed analytical method was applied to a human urine sample and 16 amino acids (His, Arg, Asn, Gln, Cit, Ser, Thr, Glu, Gly, Tyr, Ala, Trp, Val, Phe, Ile, and Leu) were quantified. The urinary amino acid concentrations were 5–960 μM. Urinary amino acid analysis using this method is expected to be clinically applicable as a novel biomarker for diseases affecting the bladder, urinary tract, and kidneys.

更新日期：2023-03-25

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