AQbD-Based UPLC-ELSD Method for Quantifying Medium Chain Triglycerides in Labrafac™ WL 1349 for Nanoemulsion Applications

Abstract

In response to recent regulatory guidelines, including ICH (International Council for Harmonisation) Q2 (R2) and Q14, we developed a UPLC-ELSD method to quantify Medium-Chain Triglycerides (MCTs) in Labrafac™ WL 1349 for nanoemulsion applications. This procedure, crafted using Analytical Quality by Design (AQbD) principles, addresses not only the validation of the methodology but also the lifecycle management challenges associated with the analysis of lipid-based excipients. Key parameters such as mobile phase composition, organic modifier, column type, flow rate, diluent, and column temperature were optimized to meet regulatory standards and ensure robustness in MCT quantification. Optimal conditions were achieved with a Waters Acquity HSS T3 (100 × 2.1 mm i.d., 1.8 μm) column at 33 °C, using a mixture of methanol (97.5%) and water (2.5%) containing 0.4% of formic acid at a flow rate of 0.41 mL/min. The method demonstrated an excellent fit on a cubic modelization for MCTs over a broad range of concentrations. Forced degradation studies, including hydrolytic (acidic and basic), oxidative, and thermal stress, confirmed the method’s suitability for possible stability scenarios. This validated UPLC method was successfully applied to quantitative analyses of bulk and formulation prototype samples containing MCTs. This AQbD-driven method enhances not only knowledge but also regulatory-compliant and cost-effective excipient control.

Introduction

LabrafacTM WL 1349 (Labrafac) is a pharmaceutical excipient composed of Medium-Chain Triglycerides (MCTs), primarily derived from caprylic (C8) and capric (C10) fatty acids. It is presented in the form of an oily liquid vehicle and is known for its excellent solubilizing properties. According to the Lipid Formulation Classification System (LFCS), Labrafac can widely contribute to formulating lipid-based drug delivery systems (LBDDS). This includes type I, type II, and type III, which are defined as oils, self-emulsifying drug delivery systems (SEDDS), and self-microemulsifying drug delivery systems (SMEDDS) [1]. Moreover, Labrafac can also play a role in formulating other kinds of lipid nanoparticles, like Nanostructured Lipid Carriers (NLC), composed of solid and liquid lipids [2]. Among these advanced formulation strategies, promising approaches like microemulsions and nanoemulsions (NEs) can be found.

Systems like NEs are particularly beneficial for enhancing the solubility and bioavailability of poorly water-soluble lipophilic drugs [3], as they are developed thanks to the addition of surfactants, capable of reducing the interfacial tension between the oil and the water phase [4]. A thermodynamically stable NE is the result of applying shear forces to the combined phases, where it is critical to select an optimal mixture of low and high hydrophilic-lipophilic balance (HLB) surfactants and lipids to guarantee the shaping of a stable NE [5,6]. Oily vehicles like Labrafac are regularly utilized in producing NEs, where their characterization consists of evaluating parameters such as droplet size, structure, and kinetical behavior. The droplet size is a significant variable that influences the stability of NEs, considering that they mostly undergo the Ostwald ripening effect, which may considerably vary the droplet size distribution and lead to emulsion polydispersity [7]. A non-homogeneous droplet size pattern inevitably results in the NE’s unpredictable stability behavior and consequent uncertain therapeutic effects.

A large-scale production line for NEs comprises multiple steps, and the methodologies can vary using high and low-energy preparation methods [8]. Given the inherited complexity of these processes, it would be considerably ambitious nowadays to encompass the stability topic just by defining droplet size interval limits at pre-defined storage conditions. The effect that the excipients’ concentration changes across time could provide on the formulation’s stability should also be addressed. The stability discussion often refers to the stability of the Active Pharmaceutical Ingredient (API) itself (e.g., regular control of related substances) in light of the drug monographs proposed by the leading regulatory framework: in the case of the NEs, it’s fundamental to provide a strict control on the excipients as well, whose concentration variations might potentially lead to unstable, and thus unsafe and inefficient pharmaceuticals [9]. Although NEs are usually reputed as stable, thanks to the lower probability of creaming, flocculation, and coalescence appearing, the potential effect of the inert ingredients blend on their stability cannot be excluded [7].

Routine control analysis of lipids has already been extensively reported in the literature [10,11,12,13], where Ultra High-Performance Liquid Chromatography (UPLC) utilizing a reversed-phase (RP) approach represents the most efficient solution time-wise and cost-wise [14]. However, the European Pharmacopeia 11.0 still proposes a method for the detection of MCT’s fatty acids composition via Gas Chromatography (GC) in the respective monograph [15]. The advantage of using HPLC-RP compared to GC lies in the possibility of avoiding the derivatization step necessary for analyzing non-volatile lipids [16]. Regarding detection, MCTs, like many triglycerides, are not easily detectable via UV mode due to the lack of chromophore groups [17]. Additionally, the absence of readily ionizable functional groups complicates MS detection. Therefore, alternative determination strategies need to be adopted to quantify lipids successfully: among these, Evaporative Light Scattering Detection (ELSD) provides excellent sensitivity and separation of lipids less volatile than the mobile phase [12]. The detector works by nebulizing the column’s effluents into a fine aerosol mist, then moving through a heated drift tube, where the mobile phase evaporates. Hence, the remaining non-volatile residues are detected based on the amount of light scattered and proportional to the amount of signal generated [18].

Nowadays, ELSD efficiently translates the necessity of having a “universal detection technique”, where the main requirement for the analyte is to be at least semi-volatile [19]. Granted the detector’s capability of identifying many different species, the method development for the quantification of MCTs contained in Labrafac should aim to sufficiently separate the different esters mixtures of caprylic (C8) and capric (C10) acids, and thus provide knowledge around the conformation and stability of the NE. Effectively, the procedure needs to target four distinct combinations of C8 and C10 [20]. An exemplary structure of Labrafac is given below in Figure 1.

Interestingly, the MCTs USP monograph does not include strict limits for the quantification of Labrafac triglyceride fatty acids, whose specification ranges lie between 50.0 and 80.0% for C8 and between 20.0 and 50.0% for C10, further justifying the necessity of regular control [15,20]. It is not excluded that the methodology could detect the remaining fatty acid derivatives from caproic, lauric, and myristic acid, as well as forms of USP’s Mono and Diglycerides [21].

The analytical constraints presented by Labrafac’s complex matrix underline the exigency of meticulous method development and validation: in this scenario, the Analytical Quality by Design (AQbD) principles offer a systematic approach to design a robust method with the desired degree of performance [22,23]. AQbD is an extension of the Quality by Design (QbD) framework defined by the European Medicines Agency (EMA), which encourages employing these principles for developing methods and manufacturing drugs with expected high-quality standards, following the ICH Q8 guideline [24,25]. Validating an analytical procedure with the AQbD approach strongly increases its reliability and consistency, providing a robust structure for critical studies like stability [26]. The first step of the AQbD workflow includes defining the Analytical Target Profile (ATP), which comprises Critical Method Attributes (CMeAs) and performance characteristics, pivotal aspects to ensure that the method fits its intended target [27]. The ATP defines the level of quality planned for the procedure, and results obtained from the chromatographic sequences should meet reasonable standards of accuracy and precision, defined by the ICH Q2 (R2) guideline [28]. The validation followed the same compendium, involving a detailed evaluation of the response, limit of detection (LOD), limit of quantification (LOQ), specificity, accuracy, precision, and robustness. As recommended in the last update of the ICH Q14 guideline, robustness and risk assessment data were acquired by using Design of Experiments (DoE), a fundamental tool to assess the relationship between CMeAs and Critical Method Parameters (CMePs), pre-selected factors that could potentially influence Labrafac’s determination [29,30]. Additionally, DoE reduces the number of runs needed to achieve the desired chromatographic conditions to fit the ATP, while it was reported to reduce out-of-trend (OOT) and out-of-specification (OOS) results [29,31]. The AQbD workflow proceeds with statistically defining the Method Operable Design Region (MODR), which is the area where the responses provided by the analytical results can be predicted within a specific range, and the method can be used [32]. Once the analytical procedure is established and validated, its lifecycle is monitored to guarantee the results’ reproducibility across time [33].

Detecting lipids constitutes a challenge in terms of retention in reversed-phase mode: the choice of mobile phase and different chromatographic parameters are critical factors to consider, and this work aims to present a robust analytical methodology capable of achieving optimal retention of the target Labrafac peaks by using solid statistical tools such as DoE and systematic approaches like the AQbD principles while defining strict boundaries for its routine use.

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Standars, samples and excipients

The standards for identification of tricaprylin ≥ 97% and tricaprin ≥ 98% were acquired by Sigma Aldrich GmbH (Vienna, Austria), meanwhile, 1,2-caprate-3-caprylate and 1,2-caprylate-3-caprate were purchased from Larodan (Solna, Sweden). The Labrafac lipophileTM WL 1349 used for the samples was kindly provided by Gattefossè SAS (Saint-Priest, France). Geleol Mono- and Diglycerides for identification and specificity were provided by Gattefossé SAS as well, while Tween® 80 was acquired from Sigma-Aldrich GmbH (Vienna, Austria).

Gaggero, A.; Marko, V.; Jeremic, D.; Tetyczka, C.; Caisse, P.; Afonso Urich, J.A. AQbD-Based UPLC-ELSD Method for Quantifying Medium Chain Triglycerides in Labrafac™ WL 1349 for Nanoemulsion Applications. Molecules 2025, 30, 486. https://doi.org/10.3390/molecules30030486

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