ABSTRACT Therapeutic oligonucleotides (ONs) have rapidly emerged as a novel class of treatments in the pharmaceutical industry, with over 25 medicines approved in the past decade. Ion‐pair reversed‐phase liquid chromatography coupled with mass spectrometry (IP‐RPLC‐MS) is widely used method for ON characterization. This review critically examines the state‐of‐the‐art developments in IP‐RPLC‐MS for ON analysis, with particular focus on the selection of optimal chromatographic conditions for three key applications: impurity profiling for single‐stranded ON, characterization of novel conjugates (e.g., lipid‐and peptide‐conjugated ONs) and double‐stranded siRNA analysis in non‐denaturing conditions. Finally, recent developments in ion‐pair‐free RPLC are discussed in the context of pharmaceutical and bioanalytical applications.
ABSTRACT Per‐ and polyfluoroalkyl substances (PFAS) are environmentally persistent contaminants that are difficult to monitor and separate because of their chemical stability and diverse physicochemical properties. In this study, the retention behavior of several PFAS, including perfluorooctane sulfonate, perfluorohexane sulfonate (PFHxS‐K), perfluorobutanesulfonate (PFBS), hexafluoropropylene oxide dimer acid, and perfluorononanoic acid, was investigated using Cyclical Electrical Field‐Flow Fractionation (CyElFFF). The selected compounds represent PFAS with different chain lengths, functional groups, and environmental relevance. Field‐off injections established the void response, while application of a cyclical electric field produced analyte‐dependent retained peaks, demonstrating that CyElFFF can induce measurable retention for multiple PFAS compounds. Voltage and frequency sweeps showed that retention behavior is sensitive to the applied electrical conditions, with increasing voltage generally increasing retention and frequency changes producing smaller but measurable changes in peak position and shape. PFBS concentration studies showed that increasing concentration primarily increased peak height, while retention ratios and peak positions changed only slightly over the tested range. The PFBS/PFHxS‐K mixture was not baseline‐resolved under the tested conditions. Because the mixture and individual standards were not matched for total PFAS concentration, conductivity, or ionic strength, the cause of the shifted mixed‐sample response could not be determined. In addition, conductivity detection could not specifically identify the contribution of each compound to the mixed profile. Overall, these results show that CyElFFF can induce and modulate retention for multiple PFAS, while baseline mixture separation will require further optimization of operating conditions and reduction of non‐ideal channel interactions. Experiments were carried out at part‐per‐million concentrations to establish fundamental transport behavior rather than to demonstrate trace‐level detection.
ABSTRACT Cassia alata flowers remain phytochemically under‐characterized relative to the leaves. However, floral tissues can harbour distinct and, in some species, more abundant phenolic and flavonoid profiles, representing an unexploited source of antioxidant phytochemicals with potential functional‐food and nutraceutical relevance. Also, Cassia alata L. (Fabaceae) is a major source of numerous anthraquinones and flavonoids, yet the secondary metabolome of its floral tissues has not yet been thoroughly studied in comparison to its vegetative parts. This research therefore aimed to examine the phytoconstituents of C. alata flower extracts and evaluate their antioxidant potential. This represents, to our knowledge, the first liquid chromatography coupled with high‐resolution tandem mass spectrometry (LC‐HRMS/MS) and 2,2‐diphenyl‐1‐picrylhydrazyl (DPPH)‐based comparative profiling of C. alata flower extracts prepared with three solvents of differing polarity. Aqueous, 70% methanol, and 70% ethanol extracts of C. alata flowers were prepared and characterized using LC‐HRMS in negative ionization mode. The antioxidant potential was determined using the DPPH radical scavenging assay. LC‐HRMS enabled the tentative identification of 42 metabolites (in aqueous, methanolic, and ethanolic extracts), which were mainly flavonoids, phenolic acids, and glycosides. Some notable metabolites determined were Quercetin, Luteolin, Isomyricitin, Kaempferol‐dihexoside, Kaempferol‐O‐rutinoside, and Sophoricoside. The DPPH assay showed that all the extracts exhibited free radical scavenging activity in a concentration‐dependent manner. The ethanolic extract exhibited the strongest activity, with the lowest IC50 value of 12.10 ± 2.22 µg/mL, followed by the aqueous extract, while the methanolic extract showed the weakest activity among the plant extracts. The observed antioxidant potential of the ethanolic extract may be associated with the wide diversity of tentatively identified flavonoid‐related metabolites. These results suggest that C. alata flower extracts may serve as potential sources of antioxidant phytochemicals for food and health‐related applications.
ABSTRACT Proteinuria, defined by an abnormally elevated concentration of proteins in the urine, serves as a critical biomarker for the early detection and diagnosis of preeclampsia. Elevated proteinuria levels adversely affect both maternal and foetal health, increasing the risk of preterm birth and low birth weight. This study focused on the identification of proteinuria in blood samples through Fourier‐transform infrared (FTIR) spectroscopy as a diagnostic tool for real‐time diagnosis. Blood plasma collected during birth was evaluated in four study groups: pregnant controls ( n = 12), pregnant women with proteinuria ( n = 13), newborn controls ( n = 12), newborns with proteinuria ( n = 14). First, attenuated total reflectance‐FTIR spectra of plasma samples were collected for subsequent spectral smoothing, vector normalisation and machine‐learning classification with various supervised machine learning algorithms, including several types of Support Vector Machine, K‐Nearest Neighbours and decision trees. We obtained (76.1 ± 3.1)% accuracy, (72.1 ± 4.8)% sensitivity and (80.4 ± 3.8)% specificity for pregnant classification, and (90.6 ± 1.5)% accuracy, (83.3 ± 2.4)% sensitivity and (98.9 ± 1.6)% specificity for newborn classification. The vibrational modes corresponding to the predominant biochemical components in blood plasma of pregnant women and newborns were listed. Sensitivity and specificity results demonstrated that FTIR can be a promising tool for early diagnosis and monitoring of individual proteinuria. Although the proposed spectroscopic approach demonstrated promising diagnostic performance, the findings should be interpreted in light of clinical conditions that could affect the biochemical profile. Further studies involving larger and more heterogeneous populations are required to validate the proposed methodology.
Phenolic and flavonoid contents in medicinal plants are essential to their growth and development and provide numerous health benefits, yet their quantification using traditional wet chemistry is labor-intensive and time-consuming. This study utilized the combination of two benchtop hyperspectral imaging (HSI) systems, namely short-wave infrared (SWIR) and visible near-infrared, for the non-destructive quantitative estimation of the phenolic and flavonoid contents present in 12 powder samples of medicinal plants. For quantitative analysis, partial least squares regression (PLSR) prediction models used several spectral preprocessing techniques. To further enhance performance, variable importance in projection (VIP) selected the most informative spectral variables and combined them with PLSR to improve predictions. The optimized VIP-PLSR models further improved the predictive performance, yielding coefficients of determination (R 2) of 0.985 and 0.996 and even lower standard root mean square error of prediction values of 0.165 and 1.150 mg/g for the respective compounds. Additionally, chemical imaging provided a clear spatial visualization of both compounds within the samples. These results underscore the potential of SWIR-HSI combined with chemometrics as an effective tool for screening and quality control in medicinal plant powders.
