Quantification of Uric Acid of Rat Serum by Liquid Chromatography-ultraviolet Detection and Its Comparison Study
10.12300/j.issn.1674-5817.2022.189
- VernacularTitle:大鼠血清尿酸液相色谱-紫外检测新方法的建立及比较研究
- Author:
Ziyin XIA
1
;
Yuanyuan CHAI
1
;
Yunxia XU
1
;
Qinwei YU
1
;
Xin HUANG
1
;
Luyong ZHANG
1
;
Zhenzhou JIANG
1
Author Information
1. New Drug Screening Center, Jiangsu Center for Pharmacodynamics Research and Evaluation, State Key Laboratory of Natural Medicines, China Pharmaceutical University, Nanjing 210009, China
- Publication Type:Journal Article
- Keywords:
Uric acid;
Liquid chromatography;
Internal standard method;
Hyperuricemia;
Rats
- From:
Laboratory Animal and Comparative Medicine
2023;43(3):314-322
- CountryChina
- Language:Chinese
-
Abstract:
ObjectiveTo establish a more accurate and sensitive liquid chromatography-ultraviolet (LC-UV) method for the determination of uric acid in rat serum, and compare the results with those of commercial kits, providing a new method for the accurate determination of uric acid in the rat hyperuricemia model induced by potassium oxonate.Methods A hyperuricemia model was established by intraperitoneal injection of potassium oxonate (300 mg/kg) into SPF-grade male SD rats, and the control group was administered an equal amount of 0.5% sodium carboxymethylcellulose solution. Blood samples were collected from the posterior orbital venous plexus and centrifuged to obtain serum samples. After precipitation with 0.1% trifluoroacetic acid-acetonitrile (containing the internal standard 3,4-dihydroxybenzylamine hydrobromide), the supernatant was injected for analysis. Uric acid was separated on a Waters XBridge HILIC column (150 mm×4.6 mm, 3.5 μm) using acetonitrile (containing 0.5% formic acid and 2 mmol/mL ammonium formate) as the organic phase and methanol solution (methanol∶water=1∶1, containing 0.5% formic acid with 2 mmol/L ammonium formate) as the aqueous phase for isocratic elution and detection at 290 nm. Serum samples treated with activated carbon were used as substitute matrices for the methodological verification. Serum uric acid levels in rats with potassium oxonate-induced hyperuricemia were measured using the established LC-UV method and commercially available kits (uricase and phosphotungstic acid methods), and the accuracies of the three methods were compared.Results Serum uric acid showed a good linear relationship (R>0.999) at mass concentration of 10–200 μg/mL in rats, the lower limit of quantification was 10 μg/mL, the accuracy ranged from -2.17% to 2.21%, the intra-batch precision ranged from 0.52% to 1.95%, the inter-batch precision ranged from 3.04% to 4.90%, and the extraction recovery ranged from 83.12% to 89.91%. In the rat model, the results obtained using the commercially available phosphotungstic acid method kit were significantly higher than those of the LC-UV method, and those obtained using the commercially available uricase method kit were significantly lower than those of the LC-UV method, but the LC-UV method showed the best recovery of the spiked sample (95.90%–99.96%).ConclusionThe LC-UV method developed in this study can determine the concentration of uric acid in rat serum with higher accuracy than commercially available kits and is recommended for the determination of serum uric acid in the rat model of hyperuricemia induced by potassium oxonate.
