Department of Chemistry, Faculty of Medicine, University of Niš, Bulevar dr Zorana Djindjica 81, 18000 Niš, Serbia; Department of Pharmacy, Faculty of Medicine, University of Niš, Bulevar dr Zorana Djindjica 81, 18000 Niš, Serbia.
J Chromatogr B Analyt Technol Biomed Life Sci. 2013 Nov 15;939:17-22. doi: 10.1016/j.jchromb.2013.08.035. Epub 2013 Sep 4.
The present paper reports the development and validation of an analytical method for doxycycline quantification in human seminal fluid by HPLC with UV detection. The separation of doxycycline was achieved at 40°C on a reversed-phase C18 column using isocratic elution. The mobile phase consisted of acetonitrile (A) and water buffered at pH 2.5 with a concentrated orthophosphoric acid (B) in the volume ratio of 20:80 (v/v), respectively. The detection was performed at 350nm. As an internal standard (IS), tetracycline was used. The proposed method involves the extraction of doxycycline from seminal fluid based on acidic precipitation of the proteins using perchloric acid. The method showed good intra- and inter-day precisions (RSD<7.0%), good accuracy (recovery for doxycycline>80%), and high correlation coefficient (r=0.998) for standards subjected to the entire procedure. The detection and quantification limits were 0.087μg/ml and 0.264μg/ml. The developed method was used to analyze doxycycline in the seminal fluids obtained from male subjects who were treated with doxycycline-hyclate. The mean doxycycline concentrations of 0.89±0.07μg/ml and 0.45±0.26μg/ml were detected in seminal fluid after 6h and 12h, respectively. This is the first study reporting extraction and HPLC determination of doxycycline in this complex sample and can be very useful in support of clinical and pharmacokinetic studies on this antibiotic.
本文报道了一种采用 HPLC-UV 法测定人精液中强力霉素浓度的分析方法的建立与验证。强力霉素在 40°C 下于反相 C18 柱上实现分离，采用等度洗脱，流动相由乙腈（A）和 pH2.5 的水（含 0.1%浓磷酸，B）以 20:80（v/v）的体积比混合而成。检测波长为 350nm。四环素被用作内标。该方法涉及基于强酸沉淀蛋白质从精液中提取强力霉素，采用高氯酸。方法日内和日间精密度（RSD<7.0%）良好，准确度（强力霉素回收率>80%）良好，标准品经全程序处理的相关系数（r=0.998）高。检测限和定量限分别为 0.087μg/ml 和 0.264μg/ml。该方法用于分析服用盐酸强力霉素的男性受试者的精液中的强力霉素。6h 和 12h 后，精液中强力霉素的平均浓度分别为 0.89±0.07μg/ml 和 0.45±0.26μg/ml。这是首次报道在这种复杂样本中提取和 HPLC 测定强力霉素，对于支持该抗生素的临床和药代动力学研究非常有用。
