Search Results (650)

The use of lecithin as an emulsifier in food supplements has increased in recent years. However, successful formation of liposomes or micelles requires an appropriate mixture of phospholipids in lecithin. To evaluate the emulsification properties of lecithin for food supplements, a reliable analytical procedure for characterizing phospholipids is necessary. A liquid chromatography–mass spectrometry method was developed to identify phospholipids in lecithin without standard reference materials. For efficient separation of phospholipids before mass spectrometric analysis, a reverse-phase high-performance liquid chromatography method was optimized using a Waters XBridge Protein BEH C4 column. The optimized chromatographic method demonstrated good linearity and precision. Molecular ions were detected in full scan mode to determine accurate mass-to-charge ratios for individual peaks in the chromatogram. A custom Python program was then used to generate a list of possible phospholipid species for each peak based on the measured mass-to-charge ratios. Tandem mass spectrometry was performed to confirm the identity of specific phospholipids by comparing experimental fragmentation patterns with theoretical predictions. Identification of the phospholipids was also confirmed with four commercially available standard reference compounds, demonstrating the reliability of the proposed approach. The developed method offers a practical and cost-effective strategy for identifying phospholipids in complex matrices, especially when standard reference compounds are unavailable. Additionally, it enables targeted selection of standard compounds for future quantitative analyses, making it a valuable tool for comprehensive lipid profiling. Full article

Reversed-phase high-performance liquid chromatography (RP-HPLC) remains one of the most widely applied analytical techniques in the development and quality control testing of finished pharmaceutical products. The combination of gradient chromatographic methods with diode-array detection (DAD) enhances selectivity, ensuring accuracy and reliability when testing drugs with diverse chemical properties in a single dosage form (i.e., fixed-dose combination (FDC) products). In this study, an RP-DAD-HPLC method was developed for the quantitative analysis of clofazimine (CFZ) and pyrazinamide (PZA) for inclusion in an FDC topical drug delivery system. Chromatographic separation was achieved using a C18 column (4.6 mm × 150 mm, 5 µm particle size) with gradient elution at 1 mL/min, employing 0.1% aqueous formic acid and acetonitrile (mobile phases). PZA and CFZ were detected at 254 nm and 284 nm, respectively. The method was validated in accordance with ICH Q2 guidelines, assessing specificity (considering interference from solvents, product matrix, and degradation products), linearity (7.8–500.0 µg/mL, r² = 0.9999), system repeatability (%RSD ≤ 2.7%), and intermediate precision (25–500 µg/mL, %RSD ≤ 0.85%). Method robustness was evaluated using a three-level Box–Behnken design (BBD) with response surface methodology (RSM) to assess the effects of variations in detection wavelength, mobile phase flow rate, and column temperature. Full article

Thin-layer chromatography (TLC) remains a widely used, cost-effective and convenient method to separate small molecules, particularly in the field of natural products and (phospho)lipids. Despite advances in chromatographic methods such as high-performance liquid chromatography (HPLC), TLC retains several advantages, including simplicity and accessibility. However, a critical step is the visualization of the separated lipids on the TLC plate. Although the majority of the regularly used methods were established decades ago, there are still a number of potential pitfalls and widely unknown aspects. This review provides a concise overview about commonly used stationary phases and the solvent systems in TLC analysis of lipids. The main focus is on visualization techniques, spanning from non-specific, destructive (charring by semi-concentrated acids) to specific, non-destructive approaches (e.g., exposition to iodine to monitor unsaturated lipids). The advantages and disadvantages of the different methods will be critically discussed and frequently occurring problems highlighted. Furthermore, the combination of TLC with mass spectrometry (MS) detection will be introduced, covering both extraction-based electrospray ionization MS techniques as well as desorption techniques such as matrix-assisted laser desorption/ionization MS. MS detection, while generally more sensitive and offering molecular specificity, introduces higher technical and financial requirements compared to conventional staining. Nonetheless, the combination of TLC with MS holds significant potential for enhancing lipidomic workflows, particularly in complex biological samples. Full article

Siraitia grosvenorii (Swingle) C. Jeffrey is widely recognized for its anti-inflammatory properties, as well as its roles in lung purification, phlegm elimination, intestinal function regulation, and anti-tumor activity. Its pharmacological activity is attributed to a diversity of functional components. However, due to the extensive application of sweet glycosides in food additives, there have been few studies on non-sweet glycosides, particularly those with high polarity. This paper investigates the chemical constituents in the non-sweet glycosides fraction of S. grosvenorii juice. First, an MCI GEL CHP20P chromatographic column was utilized to enrich the non-sweet glycosides fraction. Furthermore, two-step medium-pressure liquid chromatography (MPLC) was performed for the efficient preparative separation of high-polarity non-sweet glycosides with similar structures, using C18 and silica gel as stationary phases, respectively. Seven non-sweet glycoside compounds were identified through NMR and mass spectrometry analyses, including three new compounds (4-hydroxyphenylethanol 4-O-β-D-glucopyranosyl-(1→6)-β-D-glucopyranoside, 4-hydroxyphenylethanol 4-O-β-D-glucopyranosyl-(1→2)-β-D-glucopyranosyl-(1→6)-β-D-glucopyranoside and n-butanol 1-O-β-D-glucopyranosyl-(1→2)-β-D-glucopyranosyl-(1→6)-β-D-glucopyranoside), as well as four known ones (α-D-glucopyranosyl-(1→4)-D-glucose, α-D-glucopyranosyl-(1→2)-β-D-fructofuranoside, methoxy hydroquinone diglucoside, and β-D-glucopyranoside). The results demonstrate that mixed-mode MPLC using different stationary phases is an efficient approach for separating non-sweet glycosides from S. grosvenorii. Full article

Isousnic acid (isoUA) has been detected in a few usnic acid (UA)-producing lichens with chemotaxonomic values. IsoUA was first isolated from a specimen belonging to Cladonia arbuscula s.l. (referred to as C. mitis in the publication). However, the isolation and detection of isoUA in this Cladonia species have not been reproduced and confirmed with clear evidence. This study focused on C. arbuscula s.l. collected in Iceland and aimed to (1) identify the lichen specimen using DNA barcoding and (2) investigate whether isoUA is produced using a series of chromatographic methods. The fungal nuclear ribosomal internal transcribed spacer (nrITS) barcode was sequenced, and the specimen was identified as C. arbuscula, following recent circumscription recommendations. Routine metabolite profiling did not detect isoUA, and it could only be identified after vigorous chromatographic purification and concentration steps using flash chromatography and preparative high-performance liquid chromatography. IsoUA was found in trace quantities (~24 µg/g dry weight), which likely explains its absence in routine metabolite profiling. A rapid ultra-high-performance liquid chromatography (UHPLC) method using a pentafluorophenyl column was developed to separate UA and isoUA. Our study highlights the importance of an integrative approach combining DNA barcoding and detailed chromatographic analyses for lichen chemistry research. Full article

The main goal of this study was to develop methods for quality control of naftifine hydrochloride in solution and cream forms, focusing on “Quantitative Determination” and “Related Impurities.” New, precise, accurate, and environmentally friendly high performance liquid chromatography (HPLC) methods were developed for the determination of naftifine hydrochloride and its impurities. “Quantitative determination” was performed using a diode array detector at 254 nm with an isocratic mobile phase (1.154 g of ammonium acetate R dissolved in 300 mL of water R, followed by the addition of 0.2 mL of glacial acetic acid R, mixed well) and methanol (30:70). The chromatographic columns Gemini C18 and Luna C18 were used. “Related impurities” were separated at 270 nm using a gradient mobile phase consisting of 10 M sodium octanesulfonate, 0.4 g/L disodium hydrogen phosphate anhydrous solution (pH 6.5), acetonitrile, and the Synergi Hydro-RP chromatographic column. The developed method, validated according to ICH guidelines, showed run times of 55 min for impurity analysis and 6 min for active ingredient determination. The methods were successfully applied to the quality control of the solution and cream. Full article

Symphytum officinale L. (Boraginaceae) is a plant with proven anti-inflammatory and analgesic activity on the musculoskeletal system. The traditional use of comfrey primarily refers to its roots, although some literature also mentions the leaves as an alternative plant material. Comparing the therapeutic potential of both plant materials requires additional data on the chemical composition of S. officinale leaves and their biological properties. The aim of the study was to analyze polyphenols in comfrey leaves of different origins and to assess their antioxidant and anti-inflammatory potential against comfrey roots, also collected from different sources. Polyphenol profiles were recognized by 2D TLC and HPLC-DAD-ESI-MS methods, and quantitative analysis was performed by the HPLC-UV/Vis (high performance liquid chromatograph with-ultraviolet/visible detection) method. The antioxidant activity was assessed using DPPH (2,2-diphenyl-1-picrylhydrazyl), FRAP (ferric reducing antioxidant power), and ABTS (2,2′-azino-bis(3-ethylbenzothiazoline- 6-sulfonic acid) diammonium salt) assays, and for leaves also using the TLC-DB (thin layer chromatography-direct bioautography) technique with the DPPH radical. Chemometric analysis to assess the relationship between the antioxidant activity and the origin of comfrey plant raw materials was performed. Factor analysis (FA) confirmed that geographic origin and cultivation conditions influenced the antioxidant content of both plant raw materials. The study results indicate that comfrey leaves can substitute for comfrey roots containing not only caffeic acid derivatives but also flavonoids, and exhibiting stronger antioxidant activity. Full article

Low-flow liquid chromatography has become the primary tool for advanced chromatographic analysis and is an indispensable technique for the sensitive detection of biomolecules. In this study, we developed a new 4-tritylphenyl methacrylate-based monolithic nano-column with an internal diameter of 20 µm for bioanalytical separations in nano-liquid chromatography (nano-LC). The composition of the monolith was optimized with regard to the monomer and porogenic solvent. The column was characterized using Fourier Transformed Infrared Spectroscopy (FT-IR) spectroscopy, scanning electron microscopy (SEM) and chromatographic analyses. Chromatographic characterization was performed using homologous alkylbenzenes (ABs) and polyaromatic hydrocarbons (PAHs), which facilitate hydrophobic and π–π interactions. Run-to-run and column-to-column reproducibility values were found to be <2.51% and 2.4–3.2%, respectively. The final monolith was then used to separate six standard proteins, including β-lactoglobulin A, carbonic anhydrase, ribonuclease A (RNase A), α-chymotrypsinogen (α-chym), lysozyme (Lys), cytochrome C (Cyt C) and myoglobin (Myo), as well as three dipeptides: Alanine-tyrosine (Ala-Tyr), Glycine-phenylalanine (Gly-Phe) and L-carnosine. The nano-column was then applied to profiling peptides and proteins in the MCF-7 cell line, enabling high-resolution peptide analysis. Full article

Background: The intestinal microflora is a population of microorganisms that resides in the human gastrointestinal tract and is important in maintaining metabolic and immune homeostasis in the body. Bacteria residing in the intestine produce short-chain fatty acids (SCFAs), which communicate with, among other things, the brain–gut axis—disorders of which are one of the causes of MS-like pathologies. A particular property of SCFAs is the induction of regulatory T cells, which are finding their way into pioneering therapies for MS patients. The aim of the study is to evaluate SCFA secretion in patients with multiple sclerosis from the West Pomeranian region depending on the genotypes of rs778986 and rs3894326 polymorphisms of the FUT3 gene. Methods: The study group included 47 patients clinically diagnosed with MS. Genotyping was performed by real-time PCR using TaqMan probes. Analysis of short-chain fatty acids in faeces was performed on a quadrupole mass spectrometer coupled to a time-of-flight (QTOF) analyser coupled to an AB Sciex high-performance liquid chromatograph (UHPLC). Results: Statistical analysis did not reveal any statistically significant differences in the prevalence of the studied polymorphisms in MS patients compared to the healthy control group. It was observed that the intestinal microflora and SCFA production in MS patients may be disturbed, while the studied FUT3 gene polymorphisms probably do not have a significant effect on their concentrations. A statistical tendency towards higher caproic acid content in heterozygotes of the rs778986 polymorphism and higher valeric acid secretion in homozygotes of rs3894326 was demonstrated. Conclusions: In summary, the studied FUT3 gene polymorphisms are not overrepresented in patients with MS. The rs778986 FUT3 polymorphism may affect the caproic acid content in the faeces of patients with MS, and the rs3894326 polymorphism may affect valeric acid secretion. Due to the small sample size and sparse genotype groups, the study has limited power and negative findings may reflect Type II error; replication in larger cohorts is warranted. Full article

Biogenic amines (BAs) can be found in various foods, such as cheese, wine, and chocolate. The consumption of a sufficient quantity of BA can lead to symptoms including headaches, hypertonia, and diarrhea. For this reason, the amount of BA in food is regulated in many countries. A new method for the determination of biogenic amines in wine has been proposed, which involves derivatizing BA with p-toluene sulfonyl chloride (TsCl) and using K₂S₂O₈ to reduce the matrix effect. The derivatives of putrescine, cadaverine, histamine, and tyramine with TsCl were synthesized and characterized by ¹H NMR spectroscopy. Separation of BA derivatives was performed using a reversed-phase high-performance liquid chromatography (RP-HPLC). The chromatographic system was equipped with a reversed-phase C8 column and a diode array detector. This method was validated to analyze the above-mentioned biogenic amines simultaneously in red and white wine samples. The detection limits for putrescine, cadaverine, histamine, and tyramine in wine samples were 0.0248 mg·L⁻¹, 0.0645 mg·L⁻¹, 0.346 mg·L⁻¹ and 0.00866 mg·L⁻¹, respectively. The calibration curves showed good linearity (r > 0.999), and biogenic amines recovery varied from 83.0 to 110%. The proposed method demonstrates high sensitivity, straightforward sample preparation, and rapid analysis time. Full article

Objectives: The aim of this study was to evaluate the stability of azacitidine (AZA) under clinical storage conditions (room temperature vs. refrigeration) to identify practical protocols that minimize waste and improve cost-effectiveness. Methods: AZA solutions (1 mg/mL) were stored at 23 ± 2 °C or 4 °C. Stability was assessed using a validated high-performance liquid chromatography (HPLC) method. Chromatographic separation was achieved on a Hypersil ODS C₁₈ column (250 mm × 4.6 mm, 5 μm) using an isocratic mobile phase of 50 mM potassium phosphate buffer (pH 7.0)-acetonitrile (98:2, v/v) at a flow rate of 1.0 mL/min, with UV detection at 245 nm and a 20 μL injection volume. The method demonstrated specificity for AZA and its main degradation product (DP), with LOD and LOQ of 12.56 μg/mL and 62.8 μg/mL, respectively. Linearity (R² = 0.9928), precision (RSD% < 5 for mid/high levels), and accuracy (mean recovery 96%) were established. Results: Azacitidine degraded rapidly at room temperature, with >85% loss within 24 h. In contrast, refrigeration at 4 °C significantly delayed degradation, with only ~26% loss observed over the same 24 h period. Chromatographic analysis confirmed the formation of a primary degradation product (tentatively identified as the open-ring hydrolytic species N-(formylamidino)-N′-β-D-ribofuranosylurea based on its chromatographic behavior and literature data), consistent with the known hydrolytic pathway. The applied HPLC-UV method offered an optimal balance of specificity and practicality for monitoring this main degradation trend under clinical storage conditions, distinguishing it from more complex techniques used primarily for structural elucidation. Conclusions: The pronounced instability of reconstituted AZA underscores the critical importance of strict adherence to immediate-use protocols. Refrigeration provides only a limited stability window. Based on our kinetic data, maintaining the reconstituted solution within an acceptable degradation limit (e.g., ≤10% loss) at 4 °C would require administration within a very short timeframe, supporting current handling guidelines to ensure therapeutic efficacy and minimize economic waste. Full article

Encapsulated formulations have emerged as a promising tool for increasing nutrient absorption in the food supplement and cosmetic industries. Although the theoretical amplification factors for improving the bioavailability of encapsulated formulations are very high for poorly soluble active compounds, it has long been known that encapsulation can also enhance the absorption of water-soluble ingredients. These findings have led to the development of new technologies for encapsulating nutrients for use in the food industry. However, accurate quantification of nutrients in encapsulated formulations in the food supplement industry remains a challenge. This study presents the development and validation of novel analytical procedures for determining artemisinin in various food supplement formulations. Three formulations were prepared using different emulsifying procedures for artemisinin encapsulation. High-performance liquid chromatography with UV/Vis detection (HPLC-UV/Vis) was used for analysis. Separation was performed using a Waters ACQUITY Premier BEH C18 column. Specialized sample preparation procedures were designed to efficiently disrupt encapsulation and extract artemisinin for precise quantification. Three different sample preparation procedures were required to accurately determine the artemisinin content in the tested formulations. All methods were validated. The precision, linearity expressed as R², LOD, and LOQ of the chromatographic method were 0.39%, 0.9995, 18 µg/mL, and 26 µg/mL, respectively. Recoveries of the sample preparation methods were above 94%. The developed procedures enable accurate determination of artemisinin in encapsulated formulations, ensuring product quality and safety. These findings suggest that, for quality control of encapsulated food products, specialized analytical procedures for individual formulations may need to be developed and validated. Full article

A robust and stability-indicating Reverse Phase-High Performance Liquid Chromatography (RP-HPLC) method was developed and validated for the quantitative determination of bexagliflozin and its related impurities in accordance with the International Council for Harmonisation of Technical Requirements for Pharmaceuticals for Human Use (ICH Q2(R1)) guidelines. Chromatographic separation was achieved on a C18 column using a mobile phase of methanol and ammonium acetate buffer (pH 4.2) in a 60:40 (v/v) ratio, with a flow rate of 1.0 mL·min⁻¹ and UV detection at 220 nm. The method was validated for linearity, sensitivity (LOD and LOQ), precision, robustness, and system suitability, all within acceptable limits for low-concentration analysis. Excellent linearity (r² > 0.999) and precision (%RSD 0.3–4.4%) confirmed its reliability for stability assessment. The assay was performed at 100 µg·mL⁻¹, where all validation parameters showed %RSD values ≤ 2%, demonstrating high precision and robustness. Forced degradation studies under acidic, basic, oxidative, photolytic, and thermal conditions revealed a major degradation product formed under acidic stress. This product was isolated and structurally characterized using LC–MS, ¹H NMR, and ¹³C NMR, and is reported here for the first time. The proposed RP-HPLC method proved to be specific, precise, and reliable for the determination of bexagliflozin and its related impurities, making it suitable for routine stability testing, quality control, and pharmaceutical development applications. Full article

Infant formulas are specialized foods designed for babies and toddlers who cannot be exclusively breastfed. However, acrylamide (AA) may form during the thermal processing involved in their production. Although chromatographic techniques offer high sensitivity and detection capability for AA analysis, their application remains limited due to the complexity of diverse food matrices, high operating costs, time requirements, and environmental concerns. A new validated liquid chromatography–mass spectrometry (LC-MS) protocol for AA detection in infant formula was developed using sequential hydration, acetonitrile (ACN) precipitation, and dual-sorbent clean-up, which minimized matrix effects and ensured clarity and high reproducibility. The validated method demonstrated excellent linearity (R² = 0.9985, solvent-based; 0.9903, matrix-based), a pronounced matrix effect (−67%), satisfactory sensitivity (limit of detection, LOD: 10 µg/kg; limit of quantification, LOQ: 20 µg/kg), and consistent recovery (82–99%) with less than 15% variation. AA analysis was performed on 31 infant formula samples. The highest individual AA level (268.2 µg/kg) was detected in an amino acid-based formula intended for infants under one year of age while the highest mean concentration was found in cereal-based samples (188.1 ± 100.8 µg/kg), followed by goat’s milk-based (52.7 ± 25.67), plant-based (48.8 ± 31.68), and cow’s milk-based (27.5 ± 29.62) formulas (p < 0.001). The wide variability in AA concentrations among infant formulas can be attributed to differences in formulation, ingredient composition, manufacturing processes, and analytical methodologies. These findings highlight the need for continuous monitoring of AA levels in infant foods to ensure their safety. Full article

A rapid ultra-high-performance liquid chromatography method with diode-array (DAD) and tandem mass spectrometric (MS/MS) detection was developed and, for the first time, applied for the determination of potassium iodide in ophthalmic formulations. The approach is based on an indirect derivatization reaction in which iodide is oxidized by hydrogen peroxide to electrophilic iodine species that react with 4-hydroxybenzoic acid, forming 3-iodo-4-hydroxybenzoic acid as a stable and quantifiable product. Key reaction parameters, including oxidant concentration, temperature, and incubation time, were optimized to ensure selective mono-iodination and consistent analytical response. Chromatographic separation was performed on a C18-DE RP column using gradient elution with aqueous ammonium acetate and acetonitrile, while MS/MS detection was carried out in MRM mode under ESI(−) conditions. Both UHPLC–DAD and UHPLC–MS/MS methods were validated according to ICH Q2(R2), demonstrating linearity with r² ≥ 0.998, recovery values of 97.5–107.1%, and intraday/interday RSD values up to 3.7%. UHPLC–MS/MS provided higher sensitivity (LOD 37.7 ng/mL; LOQ 114 ng/mL), whereas UHPLC–DAD reached LOD and LOQ values of 24.9 and 75.4 µg/mL. Comparative analysis showed that DAD is suitable for routine quantification, while MS/MS allows lower detection limits and improved selectivity. The developed method offers a practical and reliable tool for the quality control of potassium iodide in ophthalmic formulations. Full article