High-throughput analysis of aqueous drug solubility, supersaturation, and aggregation behaviour using second harmonic light scattering

Abstract

Aqueous solubility is a crucial physicochemical property influencing drug absorption and bioavailability. Current solubility assays, whether assessing thermodynamic or kinetic solubility, involve trade-offs between accuracy, detection limit, speed, and resource consumption. Therefore, this study introduces a novel approach to drug solubility assessment based on non-resonant second harmonic scattering (SHS), which detects interfacial fluctuations of water molecules surrounding solutes. The apparent solubility of 14 poorly water-soluble model drugs was measured and compared to high pressure liquid chromatography (HPLC) data. Furthermore, the supersaturation propensity, defined as the ratio of solubility measured at one hour to that at 24 h, was evaluated for all 14 compounds. Lastly, the self-assembly behaviour was investigated, using sodium lauryl sulphate (SLS) as a reference system to benchmark micellization in the given forward-scattering SHS platform. The results showed a strong correlation between the SHS and HPLC solubility data (r = 0.9273). Supersaturation propensity was assessed and linked to the glass-forming ability and thermal properties of the drugs, whereby ketoconazole and tamoxifen exhibited the best supersaturation performance. Moreover, the critical micelle concentration of SLS appeared as a local minimum following a peak in SHS intensity, reflecting an increase in structural bulk centrosymmetry due to micelle formation. Similar micelle-like patterns were observed for five model drugs (i.e., amiodarone, felodipine, meclizine, tamoxifen, torcetrapib), suggesting the formation of self-assembled structures at concentrations above the solubility limit. These findings demonstrate the potential of non-resonant SHS as a promising analytical tool for solubility determination, offering a versatile, dynamic and high-throughput format with minimal compound and solvent consumption, while also providing insights into drug aggregation or self-assembly at the molecular level.

Highlights

• Second harmonics scattering (SHS) enabled fast and material-saving drug solubility testing.
• Apparent solubility values strongly correlated with HPLC-based measurements (r = 0.9273).
• Time-resolved SHS allowed for kinetic solubility profiling and detection of transient species.
• Micellization and self-assembly behaviour was studied via concentration-dependent SHS patterns.
• Method applicable to both surfactants and amphiphilic drug compounds.

Introduction

Aqueous solubility is a key physicochemical parameter that typically affects absorption and bioavailability of a drug after oral administration. Therefore, accurate solubility determination and a thorough understanding of molecular behaviour in solution are crucial from the early stages of drug development (Williams et al., 2013). Depending on the experimental configuration, solubility assays can measure either the thermodynamic (equilibrium) or kinetic solubility of a compound. State-of-the-art techniques for solubility measurement often involve trade-offs between accuracy, detection limit, speed, and resource consumption (Alsenz and Kansy, 2007).

The shake-flask method, considered the ‘gold standard’ for thermodynamic solubility determination, involves equilibrating an excess of drug in dissolution media until equilibrium is reached, followed by centrifugation and/or filtration and quantification using HPLC/MS or HPLC/UV (Barrett et al., 2022, Wyttenbach et al., 2007). While reliable and easy to perform, this method is time-consuming, requires calibration, and uses comparatively large volumes of solvent, making it less appealing from a sustainability perspective. Additionally, the challenging aspect is the addition of the drug in excess due to its sparse availability during early development. Finally, for drugs that are almost insoluble, the detection limit becomes a concern.

A second method of choice is the potentiometric acid-base titration method, introduced by Avdeef, which is used to determine intrinsic solubility and solubility-pH profiles of ionizable compounds (Avdeef, 1998, Etherson et al., 2014). This method delivers full solubility-pH profiles from a single titration, but is limited to ionizable compounds, and does not support high speed testing. Researchers have also introduced alternative solubility methods such as surface UV imaging (Boetker et al., 2011, Niederquell and Kuentz, 2014), or single particle analysis but both approaches have their limitations in providing robust quantitative data in a high-throughput (HT) format (Hokkala et al., 2022).

Current trends, especially in early discovery, lean towards HT kinetic solubility screening to rapidly evaluate a large library of compounds with minimal material consumption. Such kinetic measurements often overestimate the true equilibrium solubility due to drug supersaturation (Barrett et al., 2022). However, by extending the protocol to several hours or days, allowing enough time for the drug to reach equilibrium, the measured values closely approach the thermodynamic solubility (Sou and Bergström, 2018). Initially, compounds to be tested are dissolved in DMSO, and then dispensed in dissolution medium (the final DMSO concentration in the sample should be kept to a minimum, i.e. ≤ 1 %). There is a wide range of HT solubility assay protocols available (Alsenz and Kansy, 2007, Bevan and Lloyd, 2000, Brea et al., 2024, Dehring et al., 2004, Hoelke et al., 2009, Wyttenbach et al., 2007), however they primarily revolve around three fundamental analysis principles: (i) filtration followed by HPLC quantification, (ii) turbidimetry (measuring transmitted light) or (iii) nephelometry (measuring light scattered by suspended particles). While the limitations of HPLC have been addressed already, the benefit in speed with the turbidimetry method can be easily outweighed by the need to calibrate for each solvent used and its higher limit of detection (Alsenz and Kansy, 2007). Laser-based nephelometry, also referred to as nephelometry-based light scattering is a rapid and versatile technique for solubility measurements. However, its main drawback is a detection limit of approximately 20 µM (Hoelke et al., 2009). This implies that the amount of precipitate in the solution must be high enough to induce a light scattering intensity signal that clearly exceeds the background signal.

The present work is a pilot study to introduce a novel HT analytical technique based on non-resonant second harmonic scattering (SHS) for rapid and precise solubility determination with low resource consumption and high sensitivity. To evaluate its accuracy, the new technique was compared with state-of-the-art solubility data. The method is non-destructive, robust, and has the potential to complement the analysis with additional information on the mechanistic and kinetic aspects of drug aggregation in solution. A notable previous study demonstrated that SHS can sensitively capture the micellization processes of surfactants using a conventional cuvette-based, right-angle detection geometry (Bonhomme et al., 2021). By contrast, the present study examined whether such micellization behaviour can also be observed using a forward-scattering setup, and the approach was extended to amphiphilic drug molecules to probe their self-assembly phenomena.

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Chemicals

The chemicals used in this study have been provided from different sources. Amiodarone, tamoxifen, and torcetrapib were purchased from AK Scientific (Union City, CA, USA); corticosterone, meclizine, mefenamic acid, tartaric acid, and testosterone were purchased from Sigma-Aldrich (Merck KgaA, Taufkirchen, Germany); felodipine, fenretinide, ketoconazole, miconazole, piroxicam, and progesterone were obtained from Biosynth (Carbosynth Ltd, Compton, GB), and flurbiprofen was obtained from Acros Organics (Geel, Belgium). Sodium chloride (NaCl) and sodium lauryl sulfate (SLS) were purchased from Sigma-Aldrich (Merck KGaA, Taufkirchen, Germany). The phosphate buffer was prepared using disodium hydrogen phosphate and monopotassium phosphate, both purchased from AK Scientific (Union City, CA, USA). Anhydrous dimethyl sulfoxide (DMSO) was obtained from Sigma-Aldrich (Merck KGaA, Taufkirchen, Germany) and was used as received for stock solution preparation. Ultrapure water (conductivity ≤ 0.055 μS/cm) was obtained from a Sartorius arium® pro system (Sartorius Lab Instruments, Göttingen, Germany) and was used in all experiments. Prior to use, ultrapure water, buffer, and sodium chloride solutions were filtered through 0.22 µm syringe filters (Millex®-GS, MCE membrane, Millipore, Burlington, MA, USA).

Jitka Kalasová, Orly Tarun, Yevhen Shynkarenko, Martin Kuentz, High-throughput analysis of aqueous drug solubility, supersaturation, and aggregation behaviour using second harmonic light scattering, International Journal of Pharmaceutics, Volume 685, 2025, 126200, ISSN 0378-5173, https://doi.org/10.1016/j.ijpharm.2025.126200.

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