HPLC法测定人血浆中儿茶酚胺类物质的含量

doi:10.5246/jcps.2026.07.044

摘要/Abstract

摘要：

本研究旨在建立一种准确、高效测定人血样中酪氨酸(Tyr)、3,4-二羟基苯乙酸(DOPAC)、5-羟色胺(5-HT)、肾上腺素(E)及多巴胺(DA)的高效液相色谱分析方法，研究采用紫外检测器与电化学检测器联用技术。以香草醛为内标物，流动相为乙腈与缓冲盐溶液(25 mmol/L 醋酸钠，25 mmol/L 柠檬酸，0.01 mmol/L EDTA-2Na，2.0 mmol/L 1-辛烷磺酸钠，pH = 3.76)按体积比6:94混合，柱温为室温，流速1.0 mL/min，进样量20 μL，电化学检测器工作电压设定为800 mV，量程为10 nA。结果表明，五种分析物的线性关系良好(R²均大于0.996)，检测限(LOD)为 50–150 ng/mL，定量限(LOQ)均低于0.5 μg/mL，满足检测要求。日内精密度RSD均低于5.4%，日间精密度RSD小于15%。样本在4 °C条件下稳定性良好，提取回收率较高。该方法能够准确测定人血样中儿茶酚胺类物质的含量，具有精密度高、准确度好、稳定性强的特点，具备实际应用价值。

关键词: HPLC法, 血浆样品, 内标法, 儿茶酚胺类, 紫外-电化学检测器

Abstract:

This study was conducted to develop a robust and efficient high-performance liquid chromatography (HPLC) method for the simultaneous quantification of tyrosine (Tyr), 3,4-dihydroxyphenylacetic acid (DOPAC), serotonin (5-HT), epinephrine (E), and dopamine (DA) in human blood samples. The analytical system combined ultraviolet (UV) and electrochemical detection, with vanillin serving as the internal standard. The mobile phase comprised acetonitrile and a buffer solution (25 mmol/L sodium acetate, 25 mmol/L citric acid, 0.01 mmol/L EDTA-2Na, 2.0 mmol/L sodium 1-octanesulfonate, pH 3.76) at a volumetric ratio of 6:94. Chromatographic separation was achieved at room temperature, with a flow rate of 1.0 mL/min and an injection volume of 20 μL. The electrochemical detector was operated at 800 mV with a 10 nA range. The method exhibited excellent linearity for all five analytes (R² > 0.996), with limits of detection (LOD) ranging from 50 to 150 ng/mL and limits of quantification (LOQ) below 0.5 μg/mL, satisfying analytical requirements. Intra-day precision (RSD) was consistently below 5.4%, while inter-day precision remained under 15%. Sample stability was maintained at 4 °C, and extraction recovery rates were consistently high. This HPLC approach allowed for accurate and reliable measurement of catecholamine levels in human blood, offering high precision, satisfactory accuracy, and robust stability, underscoring its practical applicability in clinical and research settings.

Key words: HPLC method, Plasma sample, Internal standard method, Catecholamine, UV-ECD

Supporting:

阿叶门, 冉志超, 陈天萍, 潘艳飞, 刘晨曦, 蒋心惠. HPLC法测定人血浆中儿茶酚胺类物质的含量[J]. 中国药学(英文版), 2026, 35(7): 615-629.

Yemen A, Zhichao Ran, Tianping Chen, Yanfei Pan, Chenxi Liu, Xinhui Jiang. Determination of catecholamine in human plasma by a detection device of high-performance liquid chromatography[J]. Journal of Chinese Pharmaceutical Sciences, 2026, 35(7): 615-629.

图/表 19

Table 1. Gradient elution.

Table 2. Optimized gradient elution method.

Figure 1. Chromatogram of catecholamines in phosphate buffer.

Figure 2. Chromatogram of catecholamines in citrate buffer.

Figure 3. Chromatogram at an input voltage of 500 mV, high noise.

Figure 4. Chromatogram at an input voltage of 800 mV.

Table 3. Retention times for each analyte.

Figure 5. Chromatograms of 0.1 M perchloric acid (Top: UV; Bottom: ECD).

Figure 6. Chromatograms of blank rat plasma (Top: UV; Bottom: ECD).

Figure 7. Chromatograms of mixed standard solutions (Top: ECD; Bottom: UV).

Table 4. Standard curves and ranges for each analyte.

Table 5. LOD and LOQ.

Table 6. Intra-day and inter-day precision and accuracy.

Table 7. Extraction recoveries.

Table 8. Durability (RSD, %).

Table 9. Stability of samples within 10 d.

Figure 8. Chromatograms of patient blood samples (Top: UV; Bottom: ECD), Peak 1: Tyr (6.5 min); Peak 2: DOPAC (9.1 min); Peak 3: 5-HT (9.9 min); Peak 4: E (11.4 min); Peak 5: DA (15.6 min); Peak 6: Vanillin (internal standard, 16.4 min).

Table 10. Results of content determination.

Figure 9. Chromatograms under different conditions. (A) Chromatogram of 500 μg/mL E-NE mixed sample. (B) Chromatogram of blank solvent. (C) Chromatogram of internal standard solution and E-NE mixed solution. (D) Chromatogram of blank rat plasma. (E) Chromatogram of blank rat plasma and internal standard. (F) Chromatogram of blank rat plasma, internal standard and E-NE mixed solution.

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