Haginaka J, Nakagawa T, Nishino Y, Uno T
J Antibiot (Tokyo). 1981 Sep;34(9):1189-94. doi: 10.7164/antibiotics.34.1189.
An ion pair reversed phase HPLC method for the determination of clavulanic acid has been developed. Since clavulanic acid had poor absorption (lambda max 201 nm in water) in a UV-region suitable for HPLC detection, the detectability was enhanced by bathochromic shift of lambda max due to solvent effect. The shifts of lambda max were measured with the solutions containing clavulanic acid, tetrabutylammonium bromide, and phosphate buffer salts in aqueous methanol. The magnitudes of the observed shifts were investigated with respect to pH, ionic strength, methanol content, and TBAB concentration. The results indicated that TBAB concentration was the predominant factor responsible for the bathochromic shifts. Taking account of the results together with solvent effects on the retention of clavulanic acid on hydrophobic stationary phase, HPLC condition suitable for detection and separation of clavulanic acid in urine was established as follows; mobile phase: 10 mM TBAB + 0.6 mM NaH2PO4 + 0.4 mM Na2HPO4 in H2O - MeOH, 10:1 (v/v)(pH 7.02), flow rate: 1.5 ml/minute, stationary phase: LiChrosorb RP-18 (25 cm x 4.6 mm i.d.), detection: UV 220 nm. The applicability of the present method is demonstrated by determining the time course of urinary excretion of clavulanic acid after oral administration of a conjugated tablet of clavulanic acid and amoxicillin to human subject.
已开发出一种用于测定克拉维酸的离子对反相高效液相色谱法。由于克拉维酸在适用于高效液相色谱检测的紫外区域吸收较差（在水中λmax为201nm），通过溶剂效应使λmax发生红移从而提高了检测能力。用含有克拉维酸、四丁基溴化铵和磷酸盐缓冲盐的甲醇水溶液测定λmax的变化。研究了观察到的变化幅度与pH、离子强度、甲醇含量和四丁基溴化铵浓度的关系。结果表明，四丁基溴化铵浓度是导致红移的主要因素。综合考虑这些结果以及溶剂对克拉维酸在疏水固定相上保留的影响，建立了适用于检测和分离尿液中克拉维酸的高效液相色谱条件如下：流动相：10mM四丁基溴化铵 + 0.6mM NaH2PO4 + 0.4mM Na2HPO4的水 - 甲醇溶液，10:1（v/v）（pH 7.02），流速：1.5ml/分钟，固定相：LiChrosorb RP - 18（25cm×4.6mm内径），检测：紫外220nm。通过测定人体口服克拉维酸和阿莫西林复方片剂后尿液中克拉维酸排泄的时间进程，证明了本方法的适用性。
