A novel and eco-friendly reversed-phase HPLC method with fluorescence detection was developed for simultaneous estimation of two co-administered antigout drugs (lesinurad and febuxostat) with diflunisal as a nonsteroidal anti-inflammatory drug. Unlike routine methodology, the developed method was optimized using analytical quality by design approach. A full factorial design was applied to optimize the effect of variable factors on chromatographic responses. The chromatographic separation was performed using isocratic elution on the Hypersil BDS C18 column at 40°C. The mobile phase consisted of acetonitrile:potassium phosphate buffer (30.0 mM; pH 5.5, 32.2:67.8% v/v) pumped at a flow rate of 1.0 mL/min and injection volume of 20.0 μL was employed. The proposed method was able to separate the ternary mixture in <10 min. The calibration curves of diflunisal, lesinurad, and febuxostat were linear over concentration ranges of 50.0–500.0, 50.0–700.0, and 20.0–700.0 ng/mL, respectively. Recovery percentages ranging from 98.1 to 101.3% with % relative standard deviation of <2% were obtained upon spiking to human plasma samples, indicating high bioanalytical applicability. Furthermore, the method was found to be excellent green when it was assessed according to Green Analytical Procedure Index and analytical Eco-Scale guidelines.

中文翻译：

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一种新的质量设计方法，用于开发和验证绿色反相 HPLC 方法，用于同时测定 lesinurad、非布司他和二氟尼柳的荧光检测：在人血浆中的应用

开发了一种具有荧光检测的新型环保反相 HPLC 方法，用于同时评估两种联合给药的抗痛风药物（乐西乐和非布司他）与作为非甾体抗炎药的二氟尼柳。与常规方法不同，开发的方法是通过设计方法使用分析质量进行优化的。应用全因子设计来优化可变因子对色谱响应的影响。色谱分离是在 Hypersil BDS C18 柱上使用等度洗脱在 40°C 下进行的。流动相由乙腈：磷酸钾缓冲液（30.0 mM；pH 5.5，32.2：67.8% v/v）组成，以 1.0 mL/min 的流速泵入，采用 20.0 μL 的进样体积。所提出的方法能够在 <10 分钟内分离三元混合物。二氟尼柳、雷西那德和非布司他的校准曲线分别在 50.0–500.0、50.0–700.0 和 20.0–700.0 ng/mL 的浓度范围内呈线性。加标至人血浆样品后，回收率范围为 98.1% 至 101.3%，相对标准偏差 <2%，表明具有很高的生物分析适用性。此外，根据绿色分析程序指数和分析生态尺度指南评估该方法是极好的绿色。