Isoherranen N, Soback S
University of Helsinki, Department of Chemistry, Finland.
Clin Chem. 2000 Jun;46(6 Pt 1):837-42.
Gentamicin is an aminoglycoside antibiotic complex containing gentamicins C(1), C(1a), and C(2). Few methods have been described for analysis of the three gentamicin components separately in biological fluids, and none has been used in pharmacokinetic studies. Determination of the three gentamicins separately may have pharmacokinetic and toxicological implications. The present study describes development of an HPLC method for the analysis of gentamicin C(1), C(1a), and C(2) components in plasma and urine.
The three components were isolated by preparative chromatography and their identities verified by thin-layer chromatography, HPLC, mass spectrometry, nuclear magnetic resonance spectroscopy, and melting point determination. The gentamicins were extracted from the biological matrix by use of Tris buffer and polymer phase solid-phase extraction. Derivatization was carried out in the solid-phase extraction cartridge with 1-fluoro-2, 4-dinitrobenzene. The 2,4-dinitrophenyl derivatives were separated with reversed-phase HPLC and quantified by the ultraviolet absorbance at 365 nm.
The detector response was linear from the limit of quantification to 50 mg/L for the individual components. The limit of quantification was 0.07 mg/L for gentamicin C(1) and 0. 1 mg/L for gentamicins C(2) and C(1a). The recovery of the gentamicin components was 72% from plasma and 98% from urine. The method was validated for human and dog plasma and urine.
The method was repeatable and enabled the analysis of gentamicins C(1), C(1a), and C(2) in plasma and urine in concentrations covering the therapeutic range of the drug, thus being suitable for therapeutic drug monitoring and pharmacokinetic studies.
庆大霉素是一种包含庆大霉素C(1)、C(1a)和C(2)的氨基糖苷类抗生素复合物。很少有方法被描述用于在生物流体中分别分析这三种庆大霉素成分，并且没有一种方法被用于药代动力学研究。分别测定这三种庆大霉素可能具有药代动力学和毒理学意义。本研究描述了一种用于分析血浆和尿液中庆大霉素C(1)、C(1a)和C(2)成分的高效液相色谱法的开发。
通过制备色谱法分离这三种成分，并通过薄层色谱法、高效液相色谱法、质谱法、核磁共振光谱法和熔点测定法验证其身份。使用Tris缓冲液和聚合物相固相萃取从生物基质中提取庆大霉素。在固相萃取柱中用1-氟-2,4-二硝基苯进行衍生化。用反相高效液相色谱法分离2,4-二硝基苯基衍生物，并通过365nm处的紫外吸光度进行定量。
各成分的检测器响应在定量限至50mg/L范围内呈线性。庆大霉素C(1)的定量限为0.07mg/L，庆大霉素C(2)和C(1a)的定量限为0.1mg/L。庆大霉素成分从血浆中的回收率为72%，从尿液中的回收率为98%。该方法已在人和犬的血浆及尿液中得到验证。
该方法具有可重复性，能够分析血浆和尿液中庆大霉素C(1)、C(1a)和C(2)的浓度，其浓度范围涵盖了该药物的治疗范围，因此适用于治疗药物监测和药代动力学研究。
