Comparative studies of HPLC-fluorometry and LC/MS method for the determination of N-acetylneuraminic acid as a marker of deteriorated ophthalmic solutions
2011
Iwatsuka, Kinya | Yasueda, Shin-ichi | Bando, Eiji | Fujii, Hiroyuki | Terada, Takashi | Okubo, Hiroya | Iwamoto, Hiroki | Kinoshita, Mitsuhiro | Kakehi, Kazuaki
Methods for determining the deterioration of ophthalmic solutions using both high-performance liquid chromatography (HPLC) with fluorescence detection and liquid chromatography coupled with selected ion monitoring mass spectrometry (LC/MS) are described. The methods are based on the determination of N-acetylneuraminic acid (NeuAc) released by the hydrolysis of foreign bodies that contaminate eye drops during use. The released NeuAc was either labeled with 1,2-diamino-4,5-methylenedioxybenzene (DMB) for fluorometric detection or detected without derivatization by mass spectrometry. The calibration curves for NeuAc showed good linearity between 1.2ng/mL and 39ng/mL for fluorometric HPLC and 5.0ng/mL and 100ng/mL for LC/MS, respectively. Detection limits for fluorometric HPLC and LC/MS were 0.20ng/mL and 0.88ng/mL, respectively. The NeuAc content of some model glycoproteins determined by LC/MS method were 62–78% of those determined by fluorometry. The differences are attributed to matrix effects but the LC/MS method afforded sufficiently high sensitivity that NeuAc in the foreign bodies could be determined in eight of nine test samples.
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