关键词: 哇巴因, 超高效液相色谱串联质谱, 固相萃取, 酶联免疫吸附试验, 高血压

Abstract:

Objective To establish an ultra high performance liquid chromatography-tandem mass spectrometry（UPLC-MS/MS） for the determination of human serum ouabain. Methods High-specific UPLC-MS/MS was used. A deuterium-labeled ouabain-d3 was used as internal standard. Serum samples were prepared with solid phase extraction（SPE）,and a reversed phase column was used for chromatography separation. The mass spectrometer was operated in negative-ion mode with electrospray ionization（ESI） source. Methodology validation included the evaluation of matrix effect,recovery,accuracy,within-run precision,between-run precision and stability. The established UPLC-MS/MS was used to determine serum ouabain in 40 hypertension patients and 20 healthy subjects （healthy control group）. The results of UPLC-MS/MS were compared with those of enzyme-linked immunosorbent assay （ELISA）. Results The UPLC-MS/MS had a linear range from 0.02 to 5.00 ng/mL. The lower limit of quantification （LLOQ） was 0.02 ng/mL. The matrix effect of pretreatment with ABN solid phase extraction column was the smallest. The recovery rate was high （85%）. The accuracies of LLOQ,low-level （0.06 ng/mL）,middle-level （0.6 ng/mL） and high-level （4 ng/mL） quality control materials were 108.0%,89.2%,101.0% and 103.0%,respectively. The within-run coefficients of variation （CV） were 2.87%,1.95% and 0.56%,and the between-run CV were 5.98%,1.90% and 0.75%. The samples placed at room temperature for 16 h were stable,and the processed samples placed in autosampler at room temperature for 48 h were stable. Both biases were <15%. The results showed that both healthy control and hypertension groups had undetectable serum ouabain level by the established UPLC-MS/MS. The levels were 0.096 ng/mL in hypertension group and 0.062 ng/mL in healthy control group when they were determined by ELISA. Ouabain standard materials at 5 different levels （0.02,0.05,0.10,0.20 and 0.50 ng/mL） were determined by UPLC-MS/MS. It showed a positive correlation between the results and the corresponding levels with good linearity（r²>0.99）and high accuracy. The results of 5 samples determined by ELISA were very close（0.024 9-0.029 6 ng/mL）. Conclusions The level of serum ouabain is undetectable both in healthy control and hypertension groups by the established UPLC-MS/MS. There is difference for the results of serum ouabain determination between UPLC-MS/MS and ELISA.

Key words: Ouabain, Ultra high performance liquid chromatography-tandem mass spectrometry, Solid phase extraction, Enzyme-linked immunosorbent assay, Hypertension