A rapid and quantitative reversed-phase HPLC-DAD/ELSD method for lipids involved in nanoparticle formulations.

Lipid nanoparticles (LNPs) have shown great success as drug delivery systems, especially for mRNA vaccines, as those developed during the Covid-19 pandemics. Lipid analysis is critical to monitor the formulation process and control the quality of LNPs. The present study is focused on the development and validation of a high-performance liquid chromatography - diode array detector -evaporative light scattering detector (HPLC-DAD/ELSD) based method for the simultaneous quantification of 7 lipids, illustrating the main components of LNPs: ionizable lipids, the neutral co-lipid cholesterol, phospholipids, hydrophilic polymer-lipids for colloidal stability (e.g., a PEGylated lipid). In particular, this study focuses on two innovative synthetic lipids: a switchable cationic lipid (CSL3) which has demonstrated in vitro and in vivo siRNA transfection abilities, and the palmitic acid-grafted-poly(ethyloxazoline)5000 (PolyEtOx), used as an alternative polymer to address allergic reactions attributed to PEGylated lipids. The HPLC separation was achieved on a Poroshell C18 column at 50 °C using a step gradient of a mobile phase composed of water/methanol mixtures with 0.1% (v/v) trifluoroacetic acid (TFA). This method was validated following ICH Q2(R1) & (R2) guidelines in terms of linearity (R² ≥ 0.997), precision (relative standard deviation on peak areas < 5% for intermediate repeatability), accuracy (recoveries between 92.9% and 108.5%), and sensitivity. Indeed, low detection and quantitation limits were determined (between 0.02 and 0.04 µg and between 0.04 and 0.10 µg, respectively). Due to its high selectivity, this method allowed the analysis of lipid degradation products produced through degradation studies in basic, acidic, and oxidative conditions. Moreover, the method was successfully applied to the analysis of several liposome formulations at two key steps of the development process. Consequently, the reported HPLC method offers fast, versatile, selective and quantitative analysis of lipids, essential for development optimization, chemical characterization, and stability testing of LNP formulations.

An analytical study of lipid-oligonucleotide aggregation properties.

Lipid-oligonucleotides (LON) attract great interest as supramolecular scaffolds to improve the intracellular delivery of nucleic acids. Analytical characterization of LON assemblies is critical to formulation development, understanding in-vivo performance, as well as quality control. For this study, we selected LONs featuring different modifications on both oligonucleotide (with or without a G4 prone sequence) and lipid (mono or bis-alkyl chain covalently attached to the oligonucleotide sequence). Size exclusion chromatography (SEC) and, for the first time, capillary electrophoresis (CE) were investigated to study LON supramolecular self-assemblies. Results were correlated to those obtained with conventional physico-chemical characterization techniques i.e. gel electrophoresis, dynamic light scattering, and circular dichroism. In SEC, a separation between LON monomers and micelles was achieved in 5min on a TSK-gel G3000PW column at 70°C with 100% water, as mobile phase. CE conditions were optimized using a fused-silica capillary length of 10.0cm effective length at 15°C. Different background electrolytes were tested by varying the nature and the concentration of salts added. A sodium tetraborate buffer with 75mM NaCl appeared suitable to promote LON assembly. CE offers benefits to LON micelle analysis in terms of speed of analysis, high resolution, and low quantity of sample injected. Moreover, CE provides an appropriate tool to assess the impact of media of biological relevance on LON self-assembly. In this work, the key role of lipophilic tails and the formation of tetramolecular G-quadruplexes on the stability of LON micelles was confirmed.

Green reversed-phase HPLC development strategy: Application to artesunate and amodiaquine analysis.

A green analytical chemistry strategy is described to develop a reversed-phase high-performance liquid chromatography method for amodiaquine and artesunate analysis using ethanol-based mobile phases. This method development was particularly challenging due to the basicity of amodiaquine and low UV absorption of artesunate, leading to peak asymmetry and detection issues, respectively. UV detection concern was even more challenging due to the baseline drift observed with ethanol in gradient mode. Several green pH modifiers were selected for their ecofriendly character and their impact on peak shape and detection was investigated. The screening of various stationary phases (19 columns) appeared as a relevant and necessary approach to reach satisfactory peak shape of basic compounds. To support the results of this study, some additional compounds related to artesunate and amodiaquine structures were included. Methods were optimized and validated using total error approach with a mobile phase composed of ethanol and 10 mM formic acid using three different stationary phases from different manufacturers, providing flexibility of the quality control approach. Method greenness was assessed using the National Environmental Methods Index, the Green Analytical Procedure Index, and the Analytical Eco-Scale. Finally, artesunate and amodiaquine were successfully analyzed in fixed dose combination tablets.

Analysis of lipid-oligonucleotide conjugates by cyclodextrin-modified capillary zone electrophoresis.

Lipid-oligonucleotide (LONs) based bioconjugates represent an emerging class of therapeutic agents, allowing the delivery of therapeutic oligonucleotide sequences. The LON development requests accurate and efficient analytical methods. In this contribution, LON analysis methods were developed in cyclodextrin-modified capillary zone electrophoresis (CD-CZE). The LONs selected in this study feature different structures, including i) the oligonucleotide length (from 10 to 20 nucleotides), ii) the inter-nucleotide linkage chemistry (phosphodiester PDE or phosphorothioate PTO), and iii) the lipidic part: single- (LONsc) or double-chain (LONdc) lipids. In CD-CZE, the effect of several parameters on the electrophoretic peaks was investigated (buffer, CD, and capillary temperature). The binding interaction between LON and Me-ß-CD was studied in affinity capillary electrophoresis and revealed a 1:1 LON:CD complex. Non-linear regression and three usual linearization methods (y-reciprocal, x-reciprocal, and double-reciprocal) were used to determine the binding constants (K values of 2.5.104 M-1 and 2.0.104 M-1 for LON PDE and LON PTO, respectively). Quantitative methods with good performances and analysis time lower than 5 min were achieved. Importantly, the developed analysis allows a separation between the i) full-length sequence LONs and their truncated sequences, (n-1), (n-2), and (n-4)-mers and ii) LONsc, LONdc and their corresponding unconjugated oligonucleotides. This work highlights the interest of CD-CZE methods for LON analysis.

Development of a green HPLC method for the analysis of artesunate and amodiaquine impurities using Quality by Design.

Greening analytical methods has become of great interest in the field of pharmaceutical analysis to protect both the operators' health and the environment. In this work, an innovative methodology combining Quality-by-Design (QbD) and Green Chemistry principles was followed to develop a single, green and robust RP-HPLC method for the quantitative analysis of impurities of both artesunate and amodiaquine drugs. Ethanol was selected as the best ecofriendly alternative solvent in substitution to the commonly used organic solvents such as acetonitrile and methanol. To achieve method objectives, resolutions between the 10 peaks were chosen as critical method attributes (CMAs) to be optimized through QbD approach. Based on a quality risk assessment, pH, temperature, and gradient slope were then selected as critical method parameters (CMPs) and a three level full factorial design was used to model the CMAs as function of the CMPs. Response surface methodology associated to Monte Carlo simulations allowed to determine the method operable domain region (MODR), i.e., the multidimensional combination of CMPs where CMAs simultaneously satisfied specifications (Rs ≥ 1.5) with a probability at least equal to 95 %. Inside the MODR, the working point was chosen based on green criteria, involving a mobile phase composed of ethanol and 10 mM acetic acid only as pH modifier. The method was successfully validated for all impurities using accuracy profile methodology, which was fully compliant with the ICH Q2(R1) requirements. Finally, the method was applied to the analysis of amodiaquine and artesunate impurities in raw materials and formulations.

Green Analytical Methods of Antimalarial Artemether-Lumefantrine Analysis for Falsification Detection Using a Low-Cost Handled NIR Spectrometer with DD-SIMCA and Drug Quantification by HPLC.

Two green analytical approaches have been developed for the analysis of antimalarial fixed dose tablets of artemether and lumefantrine for quality control. The first approach consisted of investigating the qualitative performance of a low-cost handheld near-infrared spectrometer in combination with the principal component analysis as an exploratory tool to identify trends, similarities, and differences between pharmaceutical samples, before applying the data driven soft independent modeling of class analogy (DD-SIMCA) as a one-class classifier for proper drug falsification detection with 100% of both sensitivity and specificity in the studied cases. Despite its limited spectral range and low resolution, the handheld device allowed detecting falsified drugs with no active pharmaceutical ingredient and identifying specifically a pharmaceutical tablet brand name. The second approach was the quantitative analysis based on the green and fast RP-HPLC technique using ethanol as a green organic solvent and acetic acid as a green pH modifier. The optimal separation was achieved in 7 min using a mobile phase composed of ethanol 96% and 10 mM of acetic acid pH 3.35 (63:37, v/v). The developed method was validated according to the total error approach based on an accuracy profile, was applied to the analysis of tablets, and allowed confirming falsified drugs detected by spectroscopy.

Chromatographic study of nucleoside-lipids by RP-UHPLC-DAD/CAD.

Today, one of the most popular strategies in drug delivery is the encapsulation of therapeutic agents in supramolecular nanosystems formed from amphiphilic molecules. Synthetic nucleoside-lipids, composed of one nucleoside and lipidic chains, constitute promising new amphiphilic excipients under research in the field of pharmaceutical and biomedical applications. The aim of this work was to study the chromatographic behavior of these nucleoside-lipids in reversed-phase HPLC to establish appropriate chromatographic conditions for their analysis in drug delivery systems. The effect of the stationary phase, the organic solvent, the pH* values, and pH modifier nature of the mobile phase were studied on retention, peak shape, and detection. Good chromatographic performance was achieved on both Syncronis® C18 and Acquity® BEH C18 with mobile phases composed of MeOH/water, 95:5 (v/v) mixtures at apparent pH above 5. Dual detection by diode array detection (DAD) and charged aerosol detection (CAD) was investigated. CAD signal was found to be dependent on the type of pH modifiers added to the mobile phase. In isocratic elution, the same order of magnitude of CAD responses was obtained for the tested nucleoside-lipids. This study led to suitable chromatographic conditions for purity and stability studies of nucleoside-lipids. The purity of the synthetized molecules was established to be superior to 98%. Different stability in organic solvents was noticed depending on nucleoside-lipid structure. This first study will allow quantitative applications to establish loading ratio and encapsulation yield in future drug delivery systems composed of nucleoside-lipid-based assemblies.

Greening Reversed-Phase Liquid Chromatography Methods Using Alternative Solvents for Pharmaceutical Analysis.

The greening of analytical methods has gained increasing interest in the field of pharmaceutical analysis to reduce environmental impacts and improve the health safety of analysts. Reversed-phase high-performance liquid chromatography (RP-HPLC) is the most widely used analytical technique involved in pharmaceutical drug development and manufacturing, such as the quality control of bulk drugs and pharmaceutical formulations, as well as the analysis of drugs in biological samples. However, RP-HPLC methods commonly use large amounts of organic solvents and generate high quantities of waste to be disposed, leading to some issues in terms of ecological impact and operator safety. In this context, greening HPLC methods is becoming highly desirable. One strategy to reduce the impact of hazardous solvents is to replace classically used organic solvents (i.e., acetonitrile and methanol) with greener ones. So far, ethanol has been the most often used alternative organic solvent. Others strategies have followed, such as the use of totally aqueous mobile phases, micellar liquid chromatography, and ionic liquids. These approaches have been well developed, as they do not require equipment investments and are rather economical. This review describes and critically discusses the recent advances in greening RP-HPLC methods dedicated to pharmaceutical analysis based on the use of alternative solvents.

Nucleoside-Lipid-Based Nanocarriers for Sorafenib Delivery.

Although the application of sorafenib, a small inhibitor of tyrosine protein kinases, to cancer treatments remains a worldwide option in chemotherapy, novel strategies are needed to address the low water solubility (< 5 µM), toxicity, and side effects issues of this drug. In this context, the use of nanocarriers is currently investigated in order to overcome these drawbacks. In this contribution, we report a new type of sorafenib-based nanoparticles stabilized by hybrid nucleoside-lipids. The solid lipid nanoparticles (SLNs) showed negative or positive zeta potential values depending on the nucleoside-lipid charge. Transmission electron microscopy of sorafenib-loaded SLNs revealed parallelepiped nanoparticles of about 200 nm. Biological studies achieved on four different cell lines, including liver and breast cancers, revealed enhanced anticancer activities of Sorafenib-based SLNs compared to the free drug. Importantly, contrast phase microscopy images recorded after incubation of cancer cells in the presence of SLNs at high concentration in sorafenib (> 80 µM) revealed a total cancer cell death in all cases. These results highlight the potential of nucleoside-lipid-based SLNs as drug delivery systems.

UHPLC method for multiproduct pharmaceutical analysis by Quality-by-Design.

An innovative Analytical Quality-by-Design (AQbD) methodology was followed to develop a specific and robust UHPLC method for the simultaneous separation of 16 active pharmaceutical ingredients (APIs). In the context of pharmaceutical repositioning, these molecules have been selected as good candidates for buccal per mucous (BPM®) administration route. Given the structural and physico-chemical diversity of compounds, an innovative development strategy based on QbD was applied. The main advantage of QbD is to ensure the robustness of the method. During a first scouting phase, the C18 chromatographic column was selected. Throughout the study, acetonitrile and ethanol based-mobile phases were investigated and compared. Ethanol was chosen as an alternative to acetonitrile due to its green properties coming from its lower toxicity and sourcing from renewable sources. Screening designs were performed to identify critical process parameters (CPPs). In ethanol media, temperature turned out to be a critical factor on peak retention and separation. Response surface methodology was then carried out to optimize CPPs and define the experimental domain of the method where complete separation between all peaks was obtained. Because changes in the elution order of the compounds occurred when modifying the experimental conditions, time differences between peaks were chosen as critical quality attributes, and an original data treatment was developed. It consisted in a systematic modelling of the time intervals between all possible pairs of peaks over the whole 3D experimental domain. Finally, a desirability analysis based on the smallest predicted time interval between peaks enabled to find optimal conditions only with ethanol based-mobile phases. Optimal conditions using ethanol, a Xbridge BEH Shield RP18 column and a 500 mL starting isocratic step, were determined by maximizing the desirability value and corresponded to a gradient slope of 2.57 %/min, a pH of 4.85, and a temperature of 33.7°C. A baseline separation of the 16 APIs was achieved with resolutions superior to 2.4 and the robustness of the method was experimentally validated.

Using an innovative combination of quality-by-design and green analytical chemistry approaches for the development of a stability indicating UHPLC method in pharmaceutical products.

An innovative combination of green chemistry and quality by design (QbD) approach is presented through the development of an UHPLC method for the analysis of the main degradation products of dextromethorphan hydrobromide. QbD strategy was integrated to the field of green analytical chemistry to improve method understanding while assuring quality and minimizing environmental impacts, and analyst exposure. This analytical method was thoroughly evaluated by applying risk assessment and multivariate analysis tools. After a scouting phase aimed at selecting a suitable stationary phase and an organic solvent in accordance with green chemistry principles, quality risk assessment tools were applied to determine the critical process parameters (CPPs). The effects of the CPPs on critical quality attributes (CQAs), i.e., resolutions, efficiencies, and solvent consumption were further evaluated by means of a screening design. A response surface methodology was then carried out to model CQAs as function of the selected CPPs and the optimal separation conditions were determined through a desirability analysis. Resulting contour plots enabled to establish the design space (DS) (method operable design region) where all CQAs fulfilled the requirements. An experimental validation of the DS proved that quality within the DS was guaranteed; therefore no more robustness study was required before the validation. Finally, this UHPLC method was validated using the concept of total error and was used to analyze a pharmaceutical drug product.

Capillary and microchip electrophoretic analysis of polycyclic aromatic hydrocarbons.

Polycyclic aromatic hydrocarbons (PAHs) are ubiquitous contaminants which can reach the environment and food in different ways. Because of their high toxicity, two international regulatory institutions, the US Environmental Protection Agency and the European Food Safety Authority, have classified PAHs as priority pollutants, generating an important demand for the detection and identification of PAHs. Thus, sensitive, fast, and cheap methods for the analysis of PAHs in environmental and food samples are urgently needed. Within this context, electrophoresis, in capillary or microchip format, displays attractive features. This review presents and critically discusses the published literature on the different approaches to capillary and microchip electrophoresis analysis of PAHs.

An experimental design based strategy to optimize a capillary electrophoresis method for the separation of 19 polycyclic aromatic hydrocarbons.

Because of their high toxicity, international regulatory institutions recommend monitoring specific polycyclic aromatic hydrocarbons (PAHs) in environmental and food samples. A fast, selective and sensitive method is therefore required for their quantitation in such complex samples. This article deals with the optimization, based on an experimental design strategy, of a cyclodextrin (CD) modified capillary zone electrophoresis separation method for the simultaneous separation of 19 PAHs listed as priority pollutants. First, using a central composite design, the normalized peak-start and peak-end times were modelled as functions of the factors that most affect PAH electrophoretic behavior: the concentrations of the anionic sulfobutylether-ß-CD and neutral methyl-ß-CD, and the percentage of MeOH in the background electrolyte. Then, to circumvent computational difficulties resulting from the changes in migration order likely to occur while varying experimental conditions, an original approach based on the systematic evaluation of the time intervals between all the possible pairs of peaks was used. Finally, a desirability analysis based on the smallest time interval between two consecutive peaks and on the overall analysis time, allowed us to achieve, for the first time in CE, full resolution of all 19 PAHs in less than 18 min. Using this optimized capillary electrophoresis method, a vegetable oil was successfully analyzed, proving its suitability for real complex sample analysis.

Optimizing separation conditions of 19 polycyclic aromatic hydrocarbons by cyclodextrin-modified capillary electrophoresis and applications to edible oils.

For the first time, the separation of 19 polycyclic aromatic hydrocarbons (PAHs) listed as priority pollutants in environmental and food samples by the United States Environmental Protection Agency (US-EPA) and the European Food Safety Authority was developed in cyclodextrin (CD)-modified capillary zone electrophoresis with laser-induced fluorescence detection (excitation wavelength: 325 nm). The use of a dual CD system, involving a mixture of one neutral CD and one anionic CD, enabled to reach unique selectivity. As solutes were separated based on their differential partitioning between the two CDs, the CD relative concentrations were investigated to optimize selectivity. Separation of 19 PAHs with enhanced resolutions as compared with previous studies on the 16 US-EPA PAHs and efficiencies superior to 1.5 × 10(5) were achieved in 15 min using 10mM sulfobutyl ether-ß-CD and 20mM methyl-ß-CD. The use of an internal standard (umbelliferone) with appropriate electrolyte and sample compositions, rinse sequences and sample vial material resulted in a significant improvement in method repeatability. Typical RSD variations for 6 successive experiments were between 0.8% and 1.7% for peak migration times and between 1.2% and 4.9% for normalized corrected peak areas. LOQs in the low µg/L range were obtained. For the first time in capillary electrophoresis, applications to real vegetable oil extracts were successfully carried out using the separation method developed here.

Use of response surface methodology to optimize the simultaneous separation of eight polycyclic aromatic hydrocarbons by capillary zone electrophoresis with laser-induced fluorescence detection.

Polycyclic aromatic hydrocarbons (PAHs) are among the most targeted contaminants by international regulatory institutions. There is thus a need for fast, selective and sensitive analytical methods to quantify these compounds at trace levels in complex samples. This article focuses on the optimization by means of an experimental design of a CE method with laser-induced fluorescence detection for the fast simultaneous separation of 8 heavy PAHs among food and environmental priority pollutants: benzo(a)pyrene, benzo(a)anthracene, chrysene, benzo(b)fluoranthene, dibenzo(a,h)anthracene, indeno(1,2,3-cd)pyrene, benzo(k)fluoranthene, and benzo(ghi)perylene. In this method, capillary zone electrophoresis with a mixture of an anionic sulfobutyl ether-ß-cyclodextrin (SBE-ß-CD) and a neutral methyl-ß-cyclodextrin (Me-ß-CD) was used to separate PAHs, on the basis of their differential distribution between the two CDs. First, the factors most affecting PAH electrophoretic behavior were identified: SBE-ß-CD and Me-ß-CD concentrations and percentage of methanol added to the background electrolyte. Then, a response surface strategy using a central composite design was carried out to model the effects of the selected factors on the normalized migration times. To optimize the separation, desirability functions were applied on modeled responses: normalized migration time differences between peak end and peak start of two consecutive peaks, and overall analysis time. From the model, predicted optimum conditions were experimentally validated and full resolution of all 8 PAHs was achieved in less than 7min using a borate buffer composed of 5.3mM SBE-ß-CD, 21.5mM Me-ß-CD and 10.3% MeOH. This CE separation method was successfully applied to real edible oil analysis.