Rapid determination of amphotericin B levels in serum by high-performance liquid chromatography without interference by bilirubin

Abstract

A high-performance liquid chromatographic method for the determination of amphotericin B concentrations in human serum without bilirubin interference was developed and compared with a microbiological assay. The high-performance liquid chromatographic assay utilized a reversed-phase trimethyl silica column, simple sample preparation, and visible detection. Reproducibility studies yielded coefficient-of-variation ranges from 1.02 to 2.11% for within-day precision and 2.88 to 4.32% for between-day precision. The correlation coefficient with the microbiological assay was 0.984 for amphotericin B.Amphotericin B (AMB) is a polyene antifungal agent which was isolated from Streptomyces nodosus in 1956 (10) and remains today the drug of choice for the treatment of systemic fungal infections (5, 12). The most frequent and clinically significant side effect of AMB therapy is nephrotoxicity (8). Although toxicity is generally dose dependent, there is considerable interindividual variability among patients. Because of these large individual variations in AMB levels in serum with oral administration, a method is required for frequent and rapid estimation of drug levels which provide adequate antifungal activity but do not produce adverse effects.The concentrations of AMB in serum were previously determined by microbiological assays (2, 11). Although bioassays have been used traditionally for the determination of concentrations of antibiotics, they have limited sensitivity and reproducibility. The additional disadvantages of bioassays are their lack of specificity and the length of time required for sample analysis. High-performance liquid chromatography (HPLC) provides a sensitive and specific alternative to bioassays, with the additional advantages of high precision and rapid turnaround time. We developed a sensitive HPLC assay for the measurement of AMB in serum and compared it with the microbiological assay.The HPLC assay was performed with an LC-6A pump, an SPD-6AV UV-visible variable-wavelength detector, and a C-R4A computing integrator (Shimadzu, Kyoto, Japan). Analysis was performed on a reversed-phase CLC-trimethylsilyl column (5 ,um; 150-by 6.0-mm [inside diameter]; Shimadzu). Samples were injected with a Rheodyne syringeloading sample injector (model 7125; Shimadzu) fitted with a 50-,ul loop. The mobile phase for HPLC analysis consisted of an acetonitrile-10 mM acetate buffer (pH 7.4) mixture (40/60, vol/vol). The mobile phase was degassed and delivered with a flow of 1.0 ml/min at room temperature. The A405 of the column effluent was monitored (0.005 absorbance units full scale).Serum samples used in this study were obtained from patients with systemic fungal infections receiving AMB as well as other antibiotics for which various determinations of * Corresponding author. concentrations in serum had been ordered as part of the clinical management of the patients. Serum standards and controls were prepared by dissolving AMB (Japan Squibb, Tokyo, Japan) in dimethyl sulfoxide and diluting the solution in drug-free pool...

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].…”

Bioanalytical methods for pharmacokinetic studies of antileishmanial drugs

“…Amphotericin B has been clinically used since 1957 (Walker & Walker, 1988), at first in the treatment of fungal infections. The majority of bioanalytical assays of amphotericin B in various matrices used for human PK studies were detected using conventional UV detection (Alak et al, 1996; Ayestarán et al, 1996; Bach, 1984; Baley et al, 1990; Barco et al, 2017; Bekersky et al, 2002a; Benson & Nahata, 1989; Brassinne et al, 1987; Chabot et al, 1989; Ching et al, 1983; Cleary et al, 1997; Collette et al, 1989, 1991; Demartini et al, 2005; Egger et al, 2001; Eldem & Arican‐Cellat, 2001; Eldem et al, 2001; Ganière Monteil et al, 1998; Golas et al, 1983; Heinemann et al, 1997; Hong et al, 2007; Hope et al, 2012; Hosotsubo et al, 1988; Hideo Hosotsubo & Hosotsubo, 1989; Hülsewede & Dermoumi, 1994; Husain et al, 2010; Kobayashi et al, 1987; Koren et al, 1988; Leclercq et al, 1985; Liu et al, 1995; Lopez et al, 1998; Lopez‐Galera et al, 1995; Mayhew et al, 1983; Nath et al, 1999; Ng et al, 1996; Nilsson‐Ehle et al, 1977; Persat et al, 2009; Shihabi et al, 1988; Vogelsinger, 2006; Walsh et al, 1998; Watanabe et al, 2010; Welte et al, 2015), which was more prominent before MS detection was standardized (Al‐Quadeib et al, 2014; Bekersky et al, 2002a; Deshpande et al, 2010; Hong et al, 2007; Husain et al, 2010; Lee et al, 2001; Qin et al, 2012; Qu et al, 2017; Roseboom, Thijssen, Rosing, Alves, Sundar, et al, 2022; Schuster et al,…”

“…In most cases, sample preparation involved protein precipitation because amphotericin B is soluble in organic solvents. For total and free fraction amphotericin B quantification in plasma, serum, urine, or tissue analysis, most often simple protein precipitation was performed using methanol or acetonitrile (ACN), and sometimes with acetic acid, dimethyl sulfoxide, (EDTA), or zinc as co‐solvent for good precipitation results (Al‐Quadeib et al, 2014; Alak et al, 1996; Ayestarán et al, 1996; Baley et al, 1990; Barco et al, 2017; Bekersky et al, 2002a; Benson & Nahata, 1989; Chabot et al, 1989; Ching et al, 1983; Cleary et al, 1997; Collette et al, 1989, 1991; Demartini et al, 2005; Ganière Monteil et al, 1998; Golas et al, 1983; Heinemann et al, 1997; Hong et al, 2007; Hosotsubo et al, 1988; Hideo Hosotsubo & Hosotsubo, 1989; Brassinne et al, 1987; Hülsewede & Dermoumi, 1994; Husain et al, 2010; Koren et al, 1988; Leclercq et al, 1985; Lee et al, 2001; Lopez‐Galera et al, 1995; Lopez et al, 1998; Mayhew et al, 1983; Nath et al, 1999; Ng et al, 1996; Nilsson‐Ehle et al, 1977; Persat et al, 2009; Qin et al, 2012; Roseboom, Thijssen, Rosing, Alves, Sundar, et al, 2022; Schuster et al, 2019; Vogelsinger, 2006; Walsh et al, 1998; Watanabe et al, 2010; Welte et al, 2015). Amphotericin B in the liposomal fraction samples was primarily separated using solid‐phase extraction (SPE) to preserve the liposomal formulation of amphotericin B and separate the free fraction from the liposomal fraction for selective analysis (Deshpande et al, 2010; Egger et al, 2001; Hong et al, 2007).…”

“…In most cases, sample preparation involved protein precipitation because amphotericin B is soluble in organic solvents. For total and free fraction amphotericin B quantification in plasma, serum, urine, or tissue analysis, most often simple protein precipitation was performed using methanol or acetonitrile (ACN), and sometimes with acetic acid, dimethyl sulfoxide, (EDTA), or zinc as co-solvent for good precipitation results (Al-Quadeib et al, 2014;Alak et al, 1996;Ayestarán et al, 1996;Baley et al, 1990;Barco et al, 2017;Bekersky et al, 2002a;Benson & Nahata, 1989;Chabot et al, 1989;Ching et al, 1983;Cleary et al, 1997;Collette et al, 1989Collette et al, , 1991Demartini et al, 2005;Ganière Monteil et al, 1998;Golas et al, 1983;Heinemann et al, 1997;Hong et al, 2007;Hosotsubo et al, 1988;Hideo Hosotsubo & Hosotsubo, 1989;Brassinne et al, 1987; because of two reasons. LC-UV employs internal standards that are chemical analogues, because UV detectors cannot discriminate between isotopes and isotope-labeled amphotericin B; also, LC-MS/ MS is not commercially available on the market yet.…”

Target site bioanalysis and pharmacokinetics of antileishmanial drugs

“…Sample preparation consisted in most cases of protein precipitation because amphotericin B is soluble in organic solvents. For total and free fraction amphotericin B quantification in plasma, serum, urine or tissue analysis, most often simple protein precipitation was performed using methanol or acetonitrile, occasionally together with acetic acid, dimethyl sulfoxide (DMSO), ethylenediaminetetraacetic acid (EDTA) or zinc as co-solvent for precipitation improvements [42][43][44][47][48][49][50][51][52][53][55][56][57][59][60][61][62][63][64][65][66][67][68][69]72,73,75,[77][78][79][80][81][82][83][84][86][87][88]90]. Amphotericin B in the liposomal fraction samples were primarily prepared using solid-phase extraction (SPE) techniques to preserve the liposomal formulation of amphotericin B and separate the free fraction from the liposomal fraction for selective analysis [54,81,89] Figure 1: Chemical structures and names of antileishmanial drugs.…”