The present research paper includes the development, validation, and comparison of three analytical methods including spectrofluorimetric, ultraviolet (UV)-spectrophotometric, and spectrodensitometric methods based on high-performance thin-layer chromatographic (HPTLC) technique for the estimation of riociguat in tablet dosage form. The estimation was performed at wavelength maxima of 322 nm for UV-spectrophotometry, and for spectrofluorimetric method, excitation wavelength was 300 nm and emission wavelength was 465 nm. Quantification by HPTLC method was carried out at wavelength maxima of 323 nm using aluminum-backed pre-coated silica gel 60 F₂₅₄ as the stationary phase and ethyl acetate–methanol–glacial acetic acid (8:2:0.3, v/v) as the mobile phase. The methods were validated according to the International Council for Harmonization (ICH) Q2(R1) guideline, where the limits of detection and quantification were found to be 0.240 and 0.728 μg/mL, 0.193 and 0.586 μg/mL, and 3.35 and 10.15 ng/band for spectrofluorimetric, UV-spectrophotometric, and HPTLC method, respectively. The developed methods were successfully applied for quantification of the content of active ingredient and content uniformity. Statistical comparison was done by ANOVA and paired t test showing no significant difference between the three methods. The proposed methods were successfully applied for content uniformity test of riociguat in its tablet dosage form.

本研究论文包括三种分析方法的开发，验证和比较，包括基于高效薄层色谱（HPTLC）技术的光谱荧光法，紫外分光光度法和分光光度法，用于估计片剂中的riociguat形成。紫外分光光度法在322 nm的最大波长处进行估计，而对于分光荧光法，估计的激发波长为300 nm，发射波长为465 nm。采用铝背衬预涂硅胶60 F ₂₅₄作为固定相，并使用乙酸乙酯-甲醇-冰醋酸（8：2：0.3，v / v），通过HPTLC方法在最大波长323 nm处进行定量）作为流动相。这些方法已根据国际协调理事会（ICH）Q2（R1）指南进行了验证，发现和定量限分别为0.240和0.728μg/ mL，0.193和0.586μg/ mL，3.35和10.15 ng / band分别用于分光荧光法，紫外分光光度法和HPTLC方法。所开发的方法已成功地用于定量活性成分的含量和含量均匀性。通过ANOVA和配对t检验进行统计学比较，显示三种方法之间没有显着差异。所提出的方法已成功地应用于riociguat片剂剂型的含量均匀度测试。