title: Comparative studies of HPLC-fluorometry and LC/MS method for the determination of N-acetylneuraminic acid as a marker of deteriorated ophthalmic solutions.
abstract: 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 1.2 ng/mL and 39 ng/mL for fluorometric HPLC and 5.0 ng/mL and 100 ng/mL for LC/MS, respectively. Detection limits for fluorometric HPLC and LC/MS were 0.20 ng/mL and 0.88 ng/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.
authors: Senju Pharmaceutical Co., Ltd., 1-5-4, Murotani, Nishiku Kobe, Hyogo 651-2241, Japan. kinya-iwatsuka@senju.co.jp
authors: Kinya
authors: K
authors: Iwatsuka
authors: Shin-ichi
authors: S
authors: Yasueda
authors: Eiji
authors: E
authors: Bando
authors: Hiroyuki
authors: H
authors: Fujii
authors: Takashi
authors: T
authors: Terada
authors: Hiroya
authors: H
authors: Okubo
authors: Hiroki
authors: H
authors: Iwamoto
authors: Mitsuhiro
authors: M
authors: Kinoshita
authors: Kazuaki
authors: K
authors: Kakehi
mesh_terms: D015415:Biomarkers / Q000032:analysis; D002851:Chromatography, High Pressure Liquid / Q000379:methods*; D004340:Drug Contamination; D004847:Epithelial Cells / Q000737:chemistry; D005123:Eye / Q000378:metabolism; D005470:Fluorometry; D006023:Glycoproteins / Q000032:analysis / Q000737:chemistry; D006801:Humans; D013058:Mass Spectrometry / Q000379:methods*; D009093:Mucus / Q000737:chemistry; D019158:N-Acetylneuraminic Acid / Q000032:analysis*; D009883:Ophthalmic Solutions / Q000737:chemistry* / Q000592:standards; D010655:Phenylenediamines / Q000737:chemistry; D012680:Sensitivity and Specificity; D013666:Tears / Q000737:chemistry
chemical_list: D015415:Biomarkers; D006023:Glycoproteins; D009883:Ophthalmic Solutions; D010655:Phenylenediamines; C058886:1,2-diamino-4,5-methylenedioxybenzene; D019158:N-Acetylneuraminic Acid