Photochemical degradation of trypan blue

PurposeTo investigate the photochemical degradation of trypan blue (TB) and to identify decomposition products.MethodsDefined solution samples of TB and a mixture with lutein/zeaxanthin were exposed to blue light. Thermal degradation processes were ruled out using controls not subjected to irradiation. All samples were analyzed using optical microscopy, UV/Vis spectroscopy, matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry and nuclear magnetic resonance (NMR) spectrometry. Degradation kinetics were determined based on changes in absorbance; intermediates were identified by analyzing mass differences of characteristic fragment ion peaks within the fragmentation patterns, and assignments were verified by NMR.ResultsTB demonstrated a photochemical degradation, which can be triggered by lutein/zeaxanthin. Intermediates vary depending on the presence of lutein/zeaxanthin. The self-sensitized photodegradation of TB occurs under generation of dimethyl sulfate and presumed formation of phenol. In contrast, within the presence of lutein/zeaxanthin the decomposition of TB indicates the formation of methoxyamine and sulfonyl arin. Thermal degradation processes were not observed.ConclusionsTB demonstrated a photodegradation that may be triggered by lutein/zeaxanthin and results in the formation of cytotoxic decomposition products. Our findings contribute to understand degradation mechanisms of TB and may elucidate previous clinical and experimental observations of cellular toxicity after TB application.研究目的：探究台盼蓝（trypan blue, TB）的光化学降解行为，并鉴定其降解产物。 实验方法：将台盼蓝单一溶液样本及其与叶黄素/玉米黄质的混合溶液样本暴露于蓝光环境中。通过设置未受辐照的对照组，排除热降解过程的干扰。所有样本均采用光学显微镜法、紫外-可见（UV/Vis）光谱法、基质辅助激光解吸电离飞行时间（matrix-assisted laser desorption/ionization time-of-flight, MALDI-TOF）质谱法以及核磁共振（nuclear magnetic resonance, NMR）波谱法进行分析。降解动力学基于吸光度的变化进行测定；中间体通过分析碎裂模式下特征碎片离子峰的质量差值得以鉴定，并通过核磁共振波谱法验证其结构归属。 实验结果：台盼蓝可发生光化学降解，且该过程可被叶黄素/玉米黄质触发。降解中间体的种类取决于叶黄素/玉米黄质的存在与否。台盼蓝的自敏化光降解过程会伴随硫酸二甲酯的生成，并推测会产生苯酚。与之相反，当体系中存在叶黄素/玉米黄质时，台盼蓝的降解会生成甲氧胺与磺酰arin（sulfonyl arin）。未检测到热降解过程。 结论：台盼蓝可发生光降解反应，且该反应可被叶黄素/玉米黄质触发，最终生成具有细胞毒性的降解产物。本研究结果有助于阐明台盼蓝的降解机制，或可解释此前关于台盼蓝应用后引发细胞毒性的临床与实验观察结果。