Purpose

To develop a microwave assisted method for the rapid synthesis of A2E and also to develop a method to quantify N-retinylidene-N-retinylethanolamine(A2E), all-trans retinal dimer (ATRD), A2-glycerophospho ethanolamine (A2GPE), dihydropyridine phosphatidyl ethanolamine (A2DHPE) and monofuran A2E (MFA2E) in age matched retina.

Methods

The development of microwave assisted synthesis of A2E, its purification and characterization for its utility in quantification in human retina. The semi-quantitative method development using LC-ESI–MS, LC-ESI–MS/MS and LC-APCI-MS/MS from pooled macula and peripheral retina for the bisretinoid analysis has been done.

Results

Maximum A2E conversion using microwave assisted process took place at 80 °C for 45 min with a yield of 55.01%. Highly sensitive and specific mass spectrometric method was developed using reverse phase C-18 separation with positive electrospray ionization and positive atmospheric phase chemical ionization of tandom mass spectrometry. A gradient mobile phase separation was achieved using water and methanol with 0.1% TFA.

Multiple reaction monitoring acquisition for ESI and APCI was performed at ATRD m/z 551.2/522.2, A2GPE m/z 746.4/729.5, A2DHPEm/z 594.4/576.5, MFA2E m/z 608.2/591.2, A2E m/z 592.4/418.2. Method was validated using LC-ESI-SIM mode to determine selectivity, linearity, sensitivity, precision and accuracy.

Conclusion

An attempt towards optimization of the synthetic procedure of A2E was made so as to reduce the lengthy reaction time without compromising the yield. Developed method was capable enough for the detection of low level of bisretinids in retina.

中文翻译：

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微波辅助合成A2E和开发LC-ESI-MS方法定量人类视网膜中眼中的类视黄醇

目的

开发微波辅助快速合成A2E的方法，并开发定量N-视黄基-N-视黄基乙醇胺（A2E），全反式视网膜二聚体（ATRD），A2-甘油磷酸乙醇胺（A2GPE），二氢吡啶磷脂酰磷脂的方法年龄匹配的视网膜中的乙醇胺（A2DHPE）和单呋喃A2E（MFA2E）。

方法

微波辅助合成A2E的开发，纯化和表征，以用于人类视网膜定量。已经完成了使用LC-ESI-MS，LC-ESI-MS / MS和LC-APCI-MS / MS从合并的黄斑和周围视网膜中进行半定量方法开发的方法，用于双维生素A分析。

结果

使用微波辅助工艺的最大A2E转化率在80°C下进行45分钟，产率为55.01％。通过使用正相电喷雾正电喷雾电离和正相气相电离正离子质谱的反相C-18分离，开发了一种高灵敏度和特异性的质谱方法。使用水和含0.1％TFA的甲醇进行梯度流动相分离。

在ATRD m / z 551.2 / 522.2，A2GPE m / z 746.4 / 729.5，A2DHPE m / z 594.4 / 576.5，MFA2E m / z 608.2 / 591.2，A2E m / z 592.4 / 418.2进行ESI和APCI的多反应监测采集。该方法已使用LC-ESI-SIM模式进行了验证，以确定选择性，线性，灵敏度，精密度和准确性。

结论

尝试优化A2E的合成程序，以减少冗长的反应时间而又不影响收率。所开发的方法足以检测视网膜中低水平的维甲酸。