Quantitative extraction of amphotericin B from serum and its determination by high-pressure liquid chromatography

Abstract

Therapeutic concentrations of amphotericin B in serum were measured by reversed-phase liquid chromatography with detection at 386 nm. Complete recovery of the drug and the internal standard from a l-ml serum sample was achieved by pretreating the sample with guanidine hydrochloride and extracting it with a disposable reversed-phase phenyl extraction column. The method was sensitive to 0.005 ,ug of amphotericin B per ml and linear to at least 5 ,ug/ml. Coehicients of variation were 4.8% within-run and 6.3% between-run.Amphotericin B is an antibiotic widely used to treat systemic mycoses (10). Its use is restricted to life-threatening infections, however, because of its severe toxicity, which is usually encountered with therapeutic doses of the drug. Amphotericin B therapy therefore involves a balancing of toxic effects against antifungal activity.Until This was then mixed with 45 ml of 1% aqueous sodium dodecyl sulfate (SDS). This stock solution (100 ,ug/ml) was stored at -70°C. To make the working standard solution (1 jxg/ml), I combined 1 ml of stock solution, 1.5 ml of 1% SDS, and 97.5 ml of water (total volume, 100 ml). This solution was stored at 1-ml aliquots at -70°C.Extraction of samples. Samples (serum, aqueous standard, cerebrospinal fluid [CSF], etc.) (1 ml) were combined with 200 ,ul of ISTD solution and 2 ml of 6 M guanidine hydrochloride. After brief mixing, each sample was passed immediately through a separate, disposable phenyl extraction column ("Bond-Elut" PH, catalog no. 608101; Analytichem International, Harbor City, Calif.). Before analysis, the column was washed with one filling of methanol and then one filling of water. The sample was applied and allowed to run through, and the column was rinsed with another filling of water and allowed to drain. The drug and the ISTD were then eluted immediately from the column into a small test tube with 500 ,ul of acetonitrile-EDTA (prepared by combining 75 ml of acetonitrile with 25 ml of 5 mM disodium EDTA). SDS (1%) solution (200 ,ul) was mixed with the extract. Because of the instability of amphotericin B in the specimen after mixing with guanidine, the above steps were completed for one sample before the next sample was extracted. Amphotericin B was very stable in the final extract after SDS was added. For calibration, a 1-ml aliquot of the amphotericin B working standard solution was thawed and immediately extracted as described above.Chromatography. Chromatography was isocratic, with a solvent consisting of acetonitrile, methanol, and 5 mM EDTA (disodium salt) (187:449:354 [vol/vol]

Flow cytometric assay for estimating fungicidal activity of Amphotericin B in human serum

“…Thus, it is reasonable to assume that therapeutic regimens should aim at achieving plasma drug levels of 1-2 gg/ml. Determination of drug concentrations in serum is possible using HPLC [2,8,17,18,21,23,31,34], but this method cannot be employed for routine analyses of a large number of samples. An alternative method involving spectrophotometric determination of Amphotericin B concentrations in acetonitrile serum extracts has been reported [37].…”

Amphotericin B enzyme-linked immunosorbent assay

“…Previously, AB in spiked sera stored at 27, 4, Ϫ20, and Ϫ70ЊC was found to have shelf half-lives of 21 h at 27ЊC and 20 days at 4ЊC, but no change was found in samples frozen at Ϫ70ЊC (1). It was not discussed previously (1) whether these samples were protected from light. Therefore, on the basis of previous data, the serum samples were immediately frozen and protected from light until use.…”

Comparison of high‐performance liquid chromatography and bioassay of amphotericin B in serum

“…The Other investigators, using similar sample preparation, reported recovery rates of 98.6-100% [9], 75-80% [lo] and 53-61% [ll]. Bach [12] argued that there are at least two disadvantages to the protein precipitation technique. First, precipitation is slow and incomplete and may interfere with the assay or clog the column.…”