アミノ酸類の蛍光誘導体化試薬の開発及び生体試料中ヒドロキシプロリン、プロリン及びプロリルジペプチド類の高感度蛍光プレラベルHPLC分析

概要

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Highly sensitive fluorescent labeling reagents, 4-(5,6-dimethoxy-2-phthalimidinyl)phenylsulfonyl chloride (DPS-Cl) and 4-(5,6- dimethoxy-2-phthalimidinyl)-2-methoxyphenylsulfonyl chloride (DMS-Cl), for determination of amino acids by HPLC were developed. DPS-Cl and DMS-Cl reacted with amino acids in the basic medium to produce the corresponding fluorescent sulfonamides (DPS derivatives; Ex 315 nm, Em 385 nm: DMS derivatives; Ex 318 nm, Em 406 nm: in aqueous acetonitrile). When the reactivities of DPS-Cl and DMS-Cl were investigated by means of reversed phase HPLC using hydroxyproline (Hyp) and alanine (Ala) as model compounds, the reactions of Hyp with DPS-C1 and DMS-Cl were completed within 5 min at 25℃ and those of Ala were within 15 min at 70℃. The detection limits (signal-to-noise ratio = 3) of almost all amino acids labeled with DPS-Cl and DMS-Cl were less than 5 fmol/injection. DPS-Cl and DMS-Cl were applied to highly sensitive determination of Hyp, proline (Pro) and prolyl dipeptides in biological samples and HPLC methods for determination of free Hyp and Pro in serum, determination of total Hyp and Pro in serum and urine and determination of prolyl dipeptides, Pro and Hyp in urine were established.
福山大学の論文