Cios Agnieszka, Kuś Kamil, Szymura-Oleksiak Joanna
Department of Pharmacokinetics and Physical Pharmacy, Jagiellonian University, Collegium Medicum, 9 Medyczna St., 30-688 Kraków, Poland.
Acta Pol Pharm. 2013 Jul-Aug;70(4):631-41.
A high-performance liquid chromatographic (HPLC) method with UV and DAD detection for the quantitative determination of linezolid in human serum was developed in present work. Chromatography was carried out by reversed-phase technique on a RP-18 column with a mobile phase composed of 50 mM phosphate buffer and acetonitrile (76 : 26, v/v), adjusted to pH 3.5 with orthophosphoric acid. Serum samples were deproteinized with methanol centrifuged and then, the supernatant was analyzed using HPLC procedure. No interference was observed at the retention times of linezolid from blank serum or ten commonly used antibiotics. A concentration range from 0.50 to 30.0 g/mL was utilized to construct calibration curves. The lower limit of detection was determined to be 0.1 microg/mL of serum for both detectors. The lower limit of quantification of 0.25 microg/mL (CV = 2.6%) was established for determination using HPLC-UV and 0.5 microg/mL (CV = 5.42%) for HPLC-DAD. The recovery of linezolid was approximately 100%. Intra-day accuracy ranged from 0.97 to 12.63% and 0.74 to 10.85% for HPLC-UV and HPLC-DAD method, respectively. Intra-day precision was less than 4.69% for HPLC-UV and less than 5.42% for HPLC-DAD method. Tests confirmed the stability of linezolid in serum during three freeze-thaw cycles and during long-term storage of frozen serum for up to 6 weeks; in extracts it was stable in the HPLC autosampler over 24 h. Statistical analysis by Student's t-test showed no significant difference between the results obtained by these two methods. In summary, these methods will be used and adapted for infected patients in intensive care unit, to determine linezolid serum concentrations in order to know the pharmacokinetic profiles of linezolid.
本研究建立了一种采用紫外和二极管阵列检测的高效液相色谱（HPLC）法，用于定量测定人血清中的利奈唑胺。色谱分析采用反相技术，在RP - 18柱上进行，流动相由50 mM磷酸盐缓冲液和乙腈（76 : 26，v/v）组成，用正磷酸调节至pH 3.5。血清样品用甲醇去蛋白后离心，然后用上清液进行HPLC分析。在利奈唑胺的保留时间处，未观察到空白血清或十种常用抗生素的干扰。使用0.50至30.0 μg/mL的浓度范围构建校准曲线。两种检测器测定的血清检测下限均为0.1 μg/mL。采用HPLC - UV测定的定量下限为0.25 μg/mL（CV = 2.6%），采用HPLC - DAD测定的定量下限为0.5 μg/mL（CV = 5.42%）。利奈唑胺的回收率约为100%。HPLC - UV和HPLC - DAD方法的日内准确度分别为0.97%至12.63%和0.74%至10.85%。HPLC - UV方法的日内精密度小于4.69%，HPLC - DAD方法的日内精密度小于5.42%。试验证实利奈唑胺在血清中经过三个冻融循环以及在冷冻血清长期储存长达6周期间是稳定的；在提取物中，它在HPLC自动进样器中24小时内稳定。通过学生t检验进行的统计分析表明，这两种方法获得的结果之间无显著差异。总之，这些方法将用于并适用于重症监护病房的感染患者，以测定利奈唑胺的血清浓度，从而了解利奈唑胺的药代动力学特征。
