Chromatographia最新文献

A novel online preparative comprehensive two-dimensional liquid chromatography (2D-LC) method coupling normal-phase liquid chromatography (NPLC) and reversed-phase liquid chromatography (RPLC) has been developed using an innovative interface design. Based on a previously reported vacuum evaporation-assisted adsorption (VEAA) system, this interface simplifies the overall 2D-LC configuration by enabling the second-dimensional RPLC to share a binary pump with the NP–RP transition unit. Controlled by a special elution program, the binary pump delivers solvents for both RPLC column elution and NP–RP conversion. This integration significantly reduces system complexity. The efficacy of the system was demonstrated through preparative isolation of an ethyl acetate extract of Escherichia coli, yielding 28 high-purity compounds, thereby confirming the reliability of the NPLC × RPLC platform.

We have reported the development of a high-performance liquid chromatography (HPLC) system employing a phase-separation multiphase flow (PSMF) as the eluent—termed the phase-separation mode in HPLC. While previous studies demonstrated this system’s capability using a binary mixture of 1-naphthol and 2, 6-naphthalenedisulfonic acid, the present study extends its evaluation to a more complex sample comprising eight naphthalene derivatives. Separations were performed in the phase-separation mode at 20 °C using a biphasic eluent of water/acetonitrile/NaCl, which was one of two-phase separation mixed solutions, and three column types: two core–shell silica columns (50 mm and 150 mm) and a 150 mm triazole-modified porous silica column. Among these, the 150 mm core–shell silica column exhibited the best performance, achieving baseline separation for nearly all components at 20 °C in the phase-separation mode. The separation performance was evaluated and discussed based on the chromatographic data obtained. Furthermore, we investigated the separation mechanism in the phase-separation mode using the 150 mm core–shell silica column, considering the distribution ratios of the analytes between the two phases at 20 °C and the pH value of the eluent, both of which influence the chromatographic behavior. Collectively, these observations here supported the unique separation principle of the phase-separation mode in HPLC and its applicability to multicomponent analyte systems.

MDMB-4en-PINACA (methyl-3,3-dimethyl-2-(1-(pent-4en-1-yl)-1H-indazole-3-carbox-amido)-butanoate) and ADB-BUTINACA (N-[1-amino-3,3-dimethyl-1-oxobutan-2-yl]-1-butyl-1H-indazole-3-carboxamide) are among the recently encountered synthetic cannabinoids (SCs). MDMB-4en-PINACA was first identified in 2017, while ADB-BUTINACA emerged in 2019. These substances were initially detected by the Trabzon Council of Forensic Medicine at the end of 2023 and have since become frequently observed in regional blood and urine analyses. Although some studies have addressed the detection of these compounds in biological matrices, existing sample preparation procedures are often time-consuming and labor-intensive. This study aimed to develop and validate a rapid and reliable LC–MS/MS method for the quantification of MDMB-4en-PINACA and ADB-BUTINACA using solid-phase extraction (SPE) for sample preparation. Among the three SPE columns evaluated, OASIS HLB demonstrated the lowest matrix effect and was selected as the most suitable under the tested conditions. The method showed good sensitivity and clear chromatographic separation of both analytes. The calibration curves were linear in the range of 1.0–50 ng/mL. The limits of detection (LOD) for ADB-BUTINACA were 0.60 ng/mL in blood and 0.63 ng/mL in urine, while for MDMB-4en-PINACA, the LODs were 1.29 ng/mL and 1.33 ng/mL, respectively. No metabolites of MDMB-4en-PINACA or ADB-BUTINACA were analyzed in this study due to the unavailability of certified reference standards. Selectivity was evaluated with respect to the parent compounds and potential endogenous interferences in the biological matrices. The validated method was successfully applied to 20 real-case samples obtained from the Trabzon Council of Forensic Medicine. These findings demonstrate that the developed SPE-LC–MS/MS method is suitable for routine forensic toxicology applications involving MDMB-4en-PINACA and ADB-BUTINACA.

C18-modified bipyridinium-functionalized silica was prepared and further modified with tetras(4-sulfonatophenyl)styrene (TPE-SO₃H) via ion bonding. Thus, two novel stationary phases, including C18-modified bipyridinium stationary phase (Sil-DPC18) and TPE-SO₃H-functionalized C18-modified bipyridinium stationary phase (Sil-DPC18-TPES) were obtained. The stationary phases were characterized by elemental analysis and Fourier transform infrared spectroscopy (FT-IR). The reversed-phase chromatographic performance of Sil-DPC18 and Sil-DPC18-TPES were evaluated using the Tanaka test mixture, alkylbenzenes, and linear polycyclic aromatic hydrocarbons (PAHs). Compared to an in-house Sil-C18, Sil-DPC18-TPES and Sil-DPC18 exhibited stronger aromatic selectivity and shape selectivity, but weaker hydrophobicity and hydrophobic selectivity. Furthermore, Sil-DPC18-TPES demonstrated better hydrophobicity, hydrophobic selectivity, aromatic selectivity and shape selectivity compared to Sil-DPC18. The enhancement could be due to the introduction of TPE-SO₃H. In addition, Sil-DPC18-TPES and Sil-DPC18 exhibited good separation selectivity for PAHs, phenylesters, phenols, and phenylamines compared to Sil-C18.

Withaferin A (WFA) is a bioactive compound known for its potent anticancer, anti-inflammatory, and adaptogenic properties, predominantly derived from the medicinal plant Withania somnifera (L.) Dunal, commonly known as Ashwagandha. The concentration of WFA in field-cultivated plants exhibits significant variability in different parts and is also influenced by environmental parameters. The extraction protocols for WFA to date reveal significant variability, emphasizing the need for a standardized method to ensure accurate quantification. Hence, in the present study, we evaluated various extraction solvents, durations, pH levels, and purification methodologies to improve recovery rates and analytical precision. Our findings indicated that methanol served as the most efficient extraction solvent, with optimal extraction achieved through 25 min of sonication at neutral pH. Moreover, employing C18 solid-phase extraction with a 40 mg sorbent for 1-min reduced matrix interference. The linear range was between 2.5 and 250 ng/g of WFA, with R² of 0.999. The LOD and LOQ were 1.2 ng/g and 3.09 ng/g for the optimized analytical condition. Ultra-High-Performance Liquid Chromatography coupled with Tandem Mass Spectrometry (UHPLC–MS/MS) found WFA at 9.42 µg/g ± 6.53% in in vitro shoot cultures of Withania somnifera. In contrast, field-grown shoots had significantly higher levels at 4518.55 µg/g ± 82.07%, which may be toxic if used in formulations directly. This requires an efficient extraction protocol and estimation method. The downstream processing of field-grown shoots may be overcome using in vitro cultures grown in a controlled environment and consistent metabolite production, highlighting their potential for standardized and optimized WFA production. This method offers high-sensitivity and specificity for quantifying WFA in complex matrices, improving metabolite yield and analytical reliability for future research and standardization of plant-derived products.

Ivermectin is an antiparasitic drug used in human and animal pharmaceutical industries to treat a variety of external and internal parasites. A new reversed phase HPLC method has been developed for identification, assay, and estimation of related substances identified in bulk lots of Ivermectin drug substance. Analytes were separated on a fused-core silica particle-based Halo C18 column (150 × 4.6 mm, 2.7 µm particle size). A gradient elution at 40 °C column temperature was employed to adequately separate all analytes of interest in the samples. The new method can separate 27 Ivermectin related substances peaks including the 26-epimer of H₂B_1a from H₂B_1a peak and H₂B_1a isomer-1 peak from H₂B_1b peak which together can account for about 2.5% of peak area percentage in a typical Ivermectin API batch. The runtime of the new HPLC method is much shorter compared to the runtime of the current USP and EP methods for Ivermectin drug substance. The quantitation limit of the method is 0.10% of target analytical concentration (~ 2 mg/mL). The new method was demonstrated to be sensitive, accurate, and robust making it a (quality control) QC friendly method for routine and non-routine analysis of Ivermectin drug substance.

Esomeprazole magnesium (EMO) is the S-enantiomer of omeprazole. As a second-generation proton pump inhibitor, it is more effective than omeprazole in inhibiting gastric acid secretion. Therefore, it is of great necessity to establish a quality control method to support its pharmaceutical development. The starting materials for the preparation of EMO may contain six potential genotoxic impurities (PGIs), designated as RC16, RC17, RC18, RC20, RC21, and RC23. These impurities have similar chemical structures, all containing benzene rings or pyridine rings with small differences in substituents, which leads to similar retention behaviors in the chromatographic column, and peak overlap is likely to occur during HPLC analysis, making it impossible to accurately determine the content of impurities. To enhance the quality control, this study established a sensitive and specific HPLC–MS/MS method. Utilizing electrospray ionization (ESI) in positive multiple reaction monitoring (MRM) mode, this method was used to determine the trace amounts of six PGIs in EMO raw material. On a 40 °C Inertsil ODS-SP C18 chromatographic column (4.6 × 150 mm, 5 μm), the six PGIs were well separated in an equilibrated elution mode at a flow rate of 0.5 mL min⁻¹. 0.5% formic acid in water and methanol were used as the mobile phase A and B, respectively. The method was rigorously validated in terms of precision, accuracy, limit of detection, limit of quantification, linear range, and robustness, and with all parameters meeting pre-defined acceptance criteria.

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Capecitabine is a prodrug converted to its only active metabolite, 5-fluorouracil (5-FU), by thymidine phosphorylase. Formulations of capecitabine, such as nanocarriers and microspheres, are being developed for the treatment of breast and colorectal cancers to enhance therapeutic efficacy and improve patient compliance. The objective of this research work is to quantitatively determine capecitabine using the analytical quality by design (AQbD) methodology principle to estimate capecitabine via a Box–Behnken statistical design (BBD). Three distinct factors and three corresponding levels (mobile phase, flow rate, and injection volume) of BBD are used for method optimization and analysis of capecitabine in tablet dosage form. A rapid, highly reliable, effortless, and effective high-performance liquid chromatographic method has been developed and validated. The analysis is conducted using a Shimadzu C18 column through an isocratic elution methodology, with the mobile phase consisting of 80:20% (v/v) ethanol and water. The flow rate is set to 1.5 mL/min, with an injection volume of 10 µL and at a detection wavelength of 241 nm. Changes in the mobile phase ratio and flow rate affect the results, but the injection volume has a minimal impact. The methodology’s distinctiveness lies in its sustainable analytical framework. The developed approach demonstrated excellent greenness. The methods of greenness were expressed by the pictograms of NEMI, Modified NEMI, GAPI, Complex GAPI, AGREE, and AGREEprep, as well as the analytical eco-score value of 93 based on the greenness assessment tools. Therefore, the green method, based on the AQbD approach and validation results, is appropriate for routine analysis of capecitabine in pharmaceutical dosage forms in quality control laboratories.

Graphical Abstract

In this study, the effects of the parameters, ethanol/methanol concentration, mobile-phase pH, and column temperature, on the retention behavior of metronidazole and albendazole were investigated using a miniaturized green reverse phase liquid chromatography (green RPLC) method based on a central composite design (CCD). The multiple effects of these parameters on the retention behavior of the studied compounds were evaluated to obtain optimal liquid chromatographic separation conditions for their simultaneous determination. In contrast, the individual effects of these parameters on the studied compounds were evaluated to obtain some physicochemical constants (dissociation and thermodynamic constants). The optimum chromatographic conditions were found employing CCD and Derringer’s desirability function. Optimum analysis conditions were determined as an ethanol–water mixture containing 9.5% (v/v) ethanol with pH adjusted to 4.5 and a methanol–water mixture containing 10% methanol with pH adjusted to 4.5. The Supelco Ascentis RP Amide (150 × 4.6 mm, 2.7 µm I.D.) column was preferred for the determination of the investigated compounds in both hydro-organic mixtures. The column temperature was kept constant at 37 °C. The method developed for the simultaneous determination of the investigated compounds demonstrated a correlation coefficient of 0.999 and linearity in the concentration range of 0.5–3.0 µg/mL, with acceptable sensitivity and accuracy values in the ethanol–water hydro-organic mixture, resulting in shorter separation times. Validated according to the ICH Q2 (R1) guideline, the method was successfully applied to the tablet dosage preparation of metronidazole. The dissociation constant values (pK_a) and thermodynamic parameters ((Delta {H}^{o}), ({Delta S}^{o}) and ({Delta G}^{o})) of the compounds were estimated using the equation obtained from CCD within the limits of the selected parameters. The data obtained from this study agrees with pK_a values obtained from other analytical methods using more toxic solvents.