HSPiP and quality by design aided optimized bioanalytical method development for amphotericin B quantification from plasma samples

Abstract

Amphotericin-b (AmB) is a broad-spectrum polyene macrolide antifungal and anti-Leishmaniosis. We addressed HSPiP and QbD (quality by design) oriented HPLC (high-performance liquid chromatography) method development and validation, followed by identifying critical attributes on retention time (RT) and peak area (PA). Various quality control parameters were estimated, such as the LLOQ (lower limit of quantification) and LLOD (lower limit of detection). HSPiP and experimental solubility data were the best fit in the prediction. QbD identified acetic acid (AA) pH, column temperature, and flow rate as the prime factors having an impact on PA and RT. The optimized mobile phase included AA, acetonitrile, and methanol (05:85:10) at pH 5 fluxed into the column maintained at 30 °C. The method was sensitive, as evidenced by the low values of LLOQ (1.6 ng/mL) and LLOD (10 ng/mL) and high recovery from the plasma. The method determined pharmacokinetic parameters from the rat plasma with high accuracy, precision, robustness, sensitivity, and reproducibility.

Introduction

Amphotericin B (AmB) is a potent polyene lipophilic antifungal with high toxicity due to aggregation behavior (Tiphine et al., 1999). Moreover, it has a complicated structure, flexible conformational properties, inability to be crystallized, and two pKa values (10 and 5.7) (Faustino and Pinheiro, 2020). The reported analytical methods revealed poor sensitivity and expensive techniques to quantify AmB. Soto et al. developed HPLC methodology using a binary solvent mixture of acetonitrile and 0.005 M aqueous solution of disodium EDTA (ethylene diamine tetra AA) (30:70). However, longer run time (12.5 min) and a high linear concentration range (1 – 200 µg/mL) challenged its sensitivity to assay from the biological sample (Soto et al., 2022). AA improves the efficiency of HPLC based analysis which may be due to pH dependent solubility (pH < 5) and self-aggregation in aqueous solutions. AmB was estimated from the plasma and sputum at low quantification limit (5 ng/mL in plasma and 10 ng/mL in sputum) using a solid phase extraction (SPE) C2 cartridge (Campanero et al., 1997).

However, the SPE method was challenged with a low extraction, poor recovery, high volume of organic solvent, and poor reproducibility (Rawa-Adkonis et al., 2006). Espada et al. focused on selective analysis of AmB (from a biological sample) using the reverse phase HPLC technique (C18 column) by optimizing acetonitrile, AA, and water content in the mobile phase (52:4.3:43.7 v/v). However, longer run time (15 min) and RT (12 min) need to be re-evaluated (Espada et al., 2008). In a study, a gradient mode of HPLC (C18) was studied for the rat plasma using acetonitrile and sodium acetate buffer (10 mM) at pH 4.0 with longer run time (15 min) and RT (6.8 min) (Italia et al., 2009). Marena et al. summarized a comprehensive report on HPLC based methods of AmB analysis (Marena et al., 2022).

HSPiP (Dr. techn. Charles M. Hansen Jens Bornøsvej 16, 2970 Hørsholm, Denmark) is a predictive program to select solvents based on theoretical solubility, Hansen parameters (HSP) and RED (relative energy difference) values (Hansen, 2007). Moreover, the QbD oriented optimization process harnessed the right composition of the selected solvents and identified the impact of various prime factors on RT and PA. Lastly, the methodology was effectively applied to determine the PK (pharmacokinetics) parameters of the developed formulation after the parenteral product (Ramzan et al., 2022).

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Materials

Amphotericin B (AmB > 99 % pure) was a kind gift from Kwality Pharmaceuticals, Amritsar (Punjab) India. Soya Phospholipon 90G (P 90G) and Lipid (Compritol 888 ATO) were obtained as gift samples from Lipoid (Frigenstrabe 4, 67,065 Ludwigshafen am Rhein, Germany) and Gattefosse (Immeuble Kastle, 2 Ter Rue du Château, 92,200 Neuilly-sur-Seine, France), respectively. Solvents were purchased from S.D. Fine Chemicals Ltd. Mumbai, India. Analytical grade methanol and acetonitrile were procured from Sigma-Aldrich, Mumbai, Maharashtra, India. Surfactants and co-solvent (Tween 80 and polyethylene glycol 400) were purchased from Himedia (Mumbai, Maharashtra, India).

Mohhammad Ramzan, Tasneem Khan, Arusha Ayub, Mudassar Shahid, HSPiP and quality by design aided optimized bioanalytical method development for amphotericin B quantification from plasma samples, Journal of King Saud University – Science, Volume 36, Issue 11, 2024, 103546, ISSN 1018-3647, https://doi.org/10.1016/j.jksus.2024.103546.