关键词: 聚冠醚复合纳米纤维, 高效液相色谱, 在线固相萃取, 儿茶酚胺, 5-羟色胺, 尿

Abstract:

Biogenic monoamines, including catecholamines (CAs) and serotonin (5-HT), play critical roles in the central nervous system. They have recently been proven to be primarily useful as biomarkers for the diagnosis of CA-producing tumors. The highly polar properties of biogenic monoamines result in poor retention on conventional materials, making it challenging to simultaneously measure more biogenic monoamines from complex matrices. Moreover, the classical method of off-line pretreatment is relatively complex, labor-intensive, and incurs errors in repeatability among different operators. Therefore, the development of an on-line sample pretreatment method combined with the use of specific nanofiber adsorbents has been explored. An on-line procedure could avoid unnecessary and time-consuming steps, and enable full automation of the experimental process. In this study, an on-line packed-fiber solid-phase extraction (PFSPE) and determination method for urinary CAs (dopamine (DA), norepinephrine (NE), epinephrine (E)) and 5-HT was developed, using composite nanofibers of polycrown ether-polystyrene (PCE-PS). PCE-PS composite nanofibers prepared by electrospinning were used as adsorbents in the PFSPE column, which was connected to the on-line HPLC system. The PFSPE-HPLC equipment contained a dual ternary pump and a switching valve to enable enrichment, purification, and analysis directly in the system. The left pump was connected with the PFSPE column for sample enrichment and purification, while the right pump was attached to the analysis column for sample separation and testing. The switching valve was controlled such that after enrichment, the samples could be eluted to the analysis column for separation and detection.
The current work expands on our previous research by analyzing more target substances, and developing an on-line sample pretreatment method to simultaneously analyze four biogenic monoamines. Gradient separation aided in the satisfactory separation of the biogenic monoamines within a short retention time. The running time was set at 16 min to enable thorough enrichment, elution, and analysis. The influence of the complexing reagent (diphenylborinic acid 2-aminoethyl ester, 2 mg/mL) was also investigated with this on-line PFSPE-HPLC system. The results showed that the intensity of most analytes was significantly higher when 50 μL of the complexing reagent was added. The influence of a buffer on the extraction of the biogenic monoamines was also tested. The optimum extraction condition for the target analytes was achieved when artificial urine (AU) samples were diluted in a volume ratio of 1∶1 by phosphate- buffered saline solution (PBS, pH 7.8). Under the optimum experimental conditions, the on-line PFSPE-HPLC procedure showed good linearity (in the range of 1 ng/mL to 200 ng/mL) with correlation coefficients above 0.996 for the quantitative detection of urinary CAs (DA, NE, E) and 5-HT. For the CAs, the limit of detection (LOD) was 1 ng/mL (S/N=3), while the limit of quantitation (LOQ) was 2.5 ng/mL (S/N=10). For 5-HT, the LOD was 2.5 ng/mL (S/N=3) and the LOQ was 5 ng/mL (S/N=10). Moreover, high recovery rates and good reproducibility were obtained. The recoveries of AU and real urine spiked with CAs and 5-HT were in the range of 83.5%-117.7%, and the intra-day precision was lower than 10%. Additionally, no significant changes in the nanofibers were observed after repeated extraction, which reflected the good stability and reusability of the nanofibers. The nanofibers could be reused for more than 95 times.
The on-line PFSPE-HPLC system was successfully applied for the determination of urinary CAs and 5-HT with good precision and high sensitivity. This high level of integration and automation was significantly advantageous in terms of its repeatability, as well as reduction in the time and effort required. The proposed on-line pretreatment and determination method can provide strong technical support for the detection and diagnosis of, as well as research on related diseases in clinical practice.

Key words: polymeric crown ether composite nanofiber, high performance liquid chromatography (HPLC), on-line solid phase extraction, catecholamines, serotonin, urine