Wang L H, Smith P C, Anderson K L, Fielding R M
College of Pharmacy, University of Texas, Austin 78712.
J Chromatogr. 1992 Sep 2;579(2):259-68. doi: 10.1016/0378-4347(92)80390-c.
A sensitive and reproducible high-performance liquid chromatographic method was developed to assay ampherotericin B in plasma, blood, urine and various tissue samples. Amphotericin B was isolated from each sample matrix by solid-phase extraction (Bond-Elut). The eluate from Bond-Elut containing amphotericin B was injected onto a reversed-phase C18 column (Waters, mu Bondpak, 10 microns, 300 mm x 3.9 mm I.D.) with a mobile phase of 45% acetonitrile in 2.5 mM Na2EDTA at 1 ml/min. Detection of amphotericin B was by ultraviolet absorption at 382 nm. Blood and tissues were homogenized and extracted with methanol prior to Bond-Elut extraction. The extraction efficiencies of amphotericin B from plasma, blood and tissues were approximately 90, 70 and 75%, respectively. The sensitivity of the assay was less than or equal to 5 ng/ml for plasma, less than or equal to 25 ng/ml for blood, 2.5 ng/ml for urine and 50 ng/g for tissues. The linearity of the assay method was up to 2.5 micrograms/ml for plasma, 5 micrograms/ml for blood, 500 ng/ml for urine and 500 micrograms/g for tissues. The assay was reproducible with an intra-day coefficient of variation (C.V., n = 3) of less than 5% in general for plasma, blood and tissues. The inter-day C.V. of the assay was less than 5% for plasma (n = 5), less than 10% for blood (n = 4) and less than 5% for tissues (n = 3). The overall variability in the urine assay was generally less than 10%. This method has demonstrated significant improvement in the sensitivity and reproducibility in assaying amphotericin B in plasma and especially in blood, urine and tissues. We have employed this assay to compare the pharmacokinetic and tissue distribution profiles of amphotericin B in rats and dogs following administration of Fungizone and ABCD (amphotericin B-cholesteryl sulfate colloidal dispersion), a lipid-based dosage form. In addition, the assay method for plasma and urine samples can also be applied to pharmacokinetics studies of amphotericin B in man.
开发了一种灵敏且可重复的高效液相色谱法，用于测定血浆、血液、尿液及各种组织样本中的两性霉素B。通过固相萃取（Bond-Elut）从每个样本基质中分离出两性霉素B。将含有两性霉素B的Bond-Elut洗脱液注入反相C18柱（沃特世，μ Bondpak，10微米，300毫米×3.9毫米内径），流动相为含45%乙腈的2.5 mM Na2EDTA，流速为1毫升/分钟。通过在382纳米处的紫外吸收检测两性霉素B。在进行Bond-Elut萃取之前，将血液和组织匀浆并用甲醇萃取。两性霉素B从血浆、血液和组织中的萃取效率分别约为90%、70%和75%。该测定法的灵敏度对于血浆小于或等于5纳克/毫升，对于血液小于或等于25纳克/毫升，对于尿液为2.5纳克/毫升，对于组织为50纳克/克。该测定法的线性范围对于血浆高达2.5微克/毫升，对于血液为5微克/毫升，对于尿液为500纳克/毫升，对于组织为500微克/克。该测定法具有可重复性，血浆、血液和组织的日内变异系数（C.V. , n = 3）一般小于5%。该测定法的日间C.V. 对于血浆（n = 5）小于5%，对于血液（n = 4）小于10%，对于组织（n = 3）小于5%。尿液测定的总体变异一般小于10%。该方法在测定血浆尤其是血液、尿液和组织中的两性霉素B时，在灵敏度和可重复性方面有显著提高。我们已采用该测定法比较了大鼠和犬在给予两性霉素B去氧胆酸钠（Fungizone）和两性霉素B硫酸胆固醇胶体分散体（ABCD，一种脂质剂型）后两性霉素B的药代动力学和组织分布情况。此外，血浆和尿液样本的测定方法也可应用于人体两性霉素B的药代动力学研究。
