Determination of aspartate and glutamate in rabbit retina using polymer monolith microextraction coupled to high-performance liquid chromatography with fluorescence detection
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A simple, sensitive and low-cost method was developed for the determination of aspartate (Asp) and glutamate (Glu) in rabbit retina. Polymer monolith microextraction (PMME) using a poly(acrylamide–vinylpyridine–N,N′-methylene bisacrylamide) (AA–VP–Bis) monolithic column was combined with derivatization of Asp and Glu using 8-phenyl-(4-oxy-acetic acid N-hydroxysuccinimide ester)-4,4-difluoro-1,3,5,7-tetramethyl-4-bora-3a,4a-diaza-s-indacene (TMPAB-OSu), and this was used to analyze the derivatives of Asp and Glu by high-performance liquid chromatography (HPLC) with fluorescence detection. The conditions for the derivatization and the subsequent extraction of Asp and Glu derivatives were optimized. The enrichment factors for the derivatives of Asp and Glu were found to be 14.1 and 14.7, respectively, by PMME. The limits of detection of Asp and Glu were 0.14 and 0.53 nmol/L, respectively. The precision and recovery were evaluated with spiked retina. The inter- and intraday relative standard deviations were less than 10%. The proposed method was successfully applied to the determination of Asp and Glu levels in rabbit retina samples with different stages of intraocular hypertension.
KeywordsPolymer monolith microextraction Derivatization Excitatory amino acids 8-phenyl-(4-oxy-acetic acid N-hydroxysuccinimide ester)-4,4-difluoro-1,3,5,7-tetramethyl-4-bora-3a,4a-diaza-s-indacene (TMPAB-OSu) HPLC
This work was partly supported by grants from the National Natural Science Foundation of China (Grant No. 20475040) and the Program for New Century Excellent Talents in University (NCET-05-0616) of MOE, China.
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