Separations, Volume 7, Issue 3 (September 2020) – 13 articles

Chondroitin sulphates belong to a group of naturally occurring glycosaminoglycans and play a role in many physiological processes including ageing and the effects of various diseases. Research into chondroitin sulphates has found that the most important analytes are 4- and 6-sulphated disaccharides. We developed an HPLC method for the separation and quantification of underivatized chondroitin/dermatan sulphates—unsaturated disaccharides (4- and 6-sulphated disaccharides). This method is based on the separation of disaccharides by amido as well as amino columns under acidic conditions. These columns enabled the successful separation of 4- and 6-sulphated disaccharides using 50 (amido column) and 25 mmol/L (amino column) phosphate buffer, pH 4.25 (detection at 230 nm), at retention times of less than 10 min. The limit of quantification was 0.5 μg/mL. The applicability of this method was demonstrated through analysis of unsaturated disaccharides produced from the enzymatic digestion of chondroitin/dermatan sulphates of the solubilized extracellular matrix produced from porcine urinary bladder and human umbilical cord. Full article

(1) Background: The present investigation studies the optimization of extraction, quantification, and cytokine inhibitory effects bakuchiol (BKL) in Psoralea coryfolia Linn. (2) Methods: The seeds of Psoralea coryfolia cleaned, dried, and powdered. Different separation methods maceration, reflux, Soxhlet, and ultrasonic assisted extraction (UAE) were employed for the isolation of BKL by five pure solvents. The quantity of BKL was measured by high-performance liquid chromatography (HPLC) method to determine the highest yield percentage. The effect of optimized BKL was then tested in an animal model of sepsis induced by lipopolysaccharides (LPS). (3) Results: The UAE method was found to be the best among tested separation methods and yielded highest percentage of BKL in petroleum ether extract. Septic rats showed a significant elevation in levels of biochemical markers like AST, ALT, ALP, BIL, SCr, and BUN in plasma. Proinflammatory cytokines (TNF-α and IL-1) levels were also increased in LPS-induced animals. BKL has been found to significantly reverse these elevated levels as compared to the LPS-induced animals. (4) Conclusion: The present results suggest that BKL has positive effects when administered in animals with pathogenic shock by decreasing the circulating levels of biomarkers. Further studies are necessary to explore the clinical implications of such findings. Full article

In this paper, two new reversed-phase (RP) HPLC-UV methods enabling the quantitative achiral and chiral analysis of ruxolitinib in commercial tablets (containing 20 mg of active pharmaceutical ingredient, API) and not commercially available galenic capsules (with 5 mg of API) are described. For the achiral method based on the use of a water/acetonitrile [70:30, v/v; with 0.1% (v) formic acid] eluent, a “research validation” study was performed mostly following the “International Council for Harmonization” guidelines. All the system suitability parameters were within the acceptance criteria: tailing factor, between 1.7 and 2.0; retention factor, 2.2; number of theoretical plates, >9000. The linearity curve showed R² = 0.99 (R_xv² = 0.99), while trueness (expressed as recovery) was between 96.3 and 106.3%. Coefficient of variations (CVs) (repeatability: CV_w and intermediate precision: CV_IP) did not exceed 1.3% and 2.9%, respectively. Moreover, the use of the enantiomeric Whelk-O1 chiral stationary phases operated under similar RP eluent conditions as for the achiral analyses, and the “inverted chirality columns approach (ICCA)” allowed us to establish that the enantiomeric purity of ruxolitinib is retained upon reformulation from tablets to capsules. The two developed methods can allow accurate determinations of ruxolitinib in drug formulations for medical use. Full article

Aside from the classical residues of persistent organic pollutants (POPs), the occurrence of emerging contaminants (ECs) in the environment has become a subject of increasing concern due to their harmful impact on the aquatic environment. Wastewater treatment plant (WWTP) effluents are major sources of environmental pollution. Therefore, data concerning their existence is required. In this study, twenty compounds representative of different drug groups considered ECs and belonging to antibiotics, antipsychotics, anti-inflammatory drugs plus acesulfame K were selected to be accurately detected and quantified with UHPLC–LTQ-Orbitrap MS in hospital and urban WWTP effluents. Chromatographic parameters (column efficiency, mobile phase, etc.), as well as mass spectrometry conditions concerning ionization mode and Orbitrap analysis (ESI options, mass resolving power, AGC target, tube lens, injection time), were evaluated. Moreover, a novel fabric phase sorptive extraction (FPSE) method based on fiber glass coated with PEG300 was employed as sample preparation process. Experimental parameters affecting extraction and desorption steps such as sample pH, extraction time, ionic strength, elution time and solvent have been optimized. The optimized methodology was validated providing excellent linearity (R² > 0.99), and low detection and quantification limits up to 3.1 and 9.3 ng/L, for carbamazepine, respectively. Relative recoveries ranged from 81.1% to 114.0%, while a medium matrix effect for most of the target compounds occurred. Applying the above analytical method in effluents of WWTPs from NW Greece, nine compounds were quantified with concentrations that varied from 55.4 to 728.4 ng/L. Full article

This study presents the synthesis of a novel asymmetric 1,3-di(alkoxy)imidazolium based room temperature ionic liquid, more precisely 1-butoxy-3-ethoxy-2-ethyl-imidazolium bis(trifluoromethane)sulfonimide, and its application as an extraction solvent in liquid-phase microextraction of cannabidiol from natural cosmetics. Quantification was implemented, using a high performance liquid chromatography system coupled to ultraviolet detection. Molecular structure elucidation was performed by nuclear magnetic resonance spectroscopy. The extraction procedure was optimized by means of two different design of experiments. Additionally, a full validation was executed. The established calibration model, ranging from 0.6 to 6.0 mg g⁻¹, was linear with a coefficient of determination of 0.9993. Accuracy and precision were demonstrated on four consecutive days with a bias within −2.6 to 2.3% and a maximum relative standard deviation value of 2.5%. Recoveries, tested for low and high concentration within the calibration range, were 80%. Stability of extracted cannabidiol was proven for three days at room temperature and fourteen days at 4 °C and −20 °C. An autosampler stability for 24 h was validated. Liquid-phase microextraction of cannabidiol from different formulated cream based cosmetics was performed, including four ointments and four creams. The results show that a significantly higher selectivity could be achieved compared to a conventional extraction methods with methanol. Full article

A modified QuEChERS (quick, easy, cheap, effective, rugged, and safe) sample preparation method coupled with liquid chromatography–tandem mass spectrometry (LC–MS/MS) was proposed for the determination of α-, β-, and γ-hexabromocyclododecane (HBCD) diastereomers in whole-fish homogenate samples. The main modification of the QuEChERS method concerned a clean-up step in which the combination of pH-tuned dispersive liquid–liquid microextraction (DLLME) with 18.4 M H₂SO₄ digestion allowed to successfully eliminate the matrix substances from the final extract. For the target HBCDs, good response linearity was obtained with coefficients of determination (R²) >0.998 for the concentration range corresponding to 0.1–50 ng of analyte per g of sample. Limits of quantifications (LOQs) were 0.15–0.25 ng g⁻¹ ww (wet weight), and the recoveries from samples spiked at levels of 0.5 and 5 ng g⁻¹ ranged from 89% to 102% with relative standard deviations <7.5%. The accuracy of the method was verified by analysis of the NIST standard reference material SRM 1947, and a good agreement (90%) was obtained with the certified value for the α-HBCD present in the sample. Finally, the method was applied to the analysis of 293 fish samples collected in water bodies from all over Slovakia, in which the highest concentrations were determined for α-HBCD with the maximum value of 31 ng g⁻¹ ww. Full article

The cytotoxic effects of the crude extract of Loranthus acaciae Zucc. and its n-hexane, chloroform, and n-butanol fractions were assessed against three cancer cell lines using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide assay. Cell apoptosis was determined using an annexin V-phycoerythrin/7-aminoactinomycin kit. We observed that the L. acaciae n-hexane extract (LAHE) could inhibit cancer cell growth, particularly of MCF7 and A549 cells. Chromatographic purification of LAHE and nuclear magnetic resonance analysis led to the identification of two compounds from this plant species, namely, betulinic acid and β-sitosterol, for the first time. Flow cytometry study suggested that betulinic acid induced cell death via apoptosis, as a distinguished marked enhancement in the early and late apoptosis of human lung (A549) and breast (MCF-7) cancer cell lines. The isolated compounds were further estimated concurrently in LAHE using a validated high-performance thin-layer chromatographic (HPTLC) method on a 10 × 10 cm² HPTLC plate with chloroform, methanol, and glacial acetic acid (97:2:1, v/v/v) as the mobile phase and a λ_max of 540 nm. The amounts of betulinic acid and β-sitosterol in LAHE were 69.46 and 135.53 µg/mg of dried weight of extract, respectively. The excellent cytotoxic effect of LAHE could be attributed to the presence of ample amounts of betulinic acid. Full article

Methylation of one hydroxyl group of monosaccharides occurs in some bacteria, fungi, worms, molluscs, and also in plants. Although knowledge on the exact functions of this process is missing, methylation is an option to modulate glycan structures thereby leading to new biological activities. In plants, methylated monosaccharides are often present in minor amounts and, therefore, overseen in analytical investigations. A special difficulty is the distinction between 3-O-methyl- and 4-O-methyl-hexoses, due to similar fragmentation patterns of methylated alditol acetates in gas-chromatography with mass spectrometric detection and, in the case of galactose, identical retention times due to symmetry. We, therefore, developed and validated an easy method for the quantitative distinction between 3-O-methyl- and 4-O-methyl-hexoses and showed its functionality by quantification of 3-O-methyl galactose in a high molecular weight polysaccharide mixture from the charophyte Spirogyra. A systematic search for methylated monosaccharides in different plant lineages may offer new insights in plant cell wall evolution. Full article

In this study, we evaluate the use of various two-dimensional liquid chromatographic methods to characterize water-soluble, synthetically grafted bio-polymers, consisting of long poly(acrylic acid) chains and short maltodextrin grafts. The confirmation of the presence of grafting and the estimation of its extent is challenging. It is complicated by the limited solubility of polymers, their structural dispersity and chemical heterogeneity. Moreover, the starting materials (and other reagents, reaction products and additives) may be present in the product. Reversed-phase liquid chromatography (RPLC), hydrophilic-interaction liquid chromatography (HILIC) and size-exclusion chromatography (SEC) were used to characterize the product, as well as the starting materials. Additionally, fractions were collected for off-line characterization by infrared spectroscopy and mass spectrometry. The one-dimensional separation methods were found to be inconclusive regarding the grafting question. Breakthrough (the early elution of polymer fractions due to strong injection solvents) is shown to be a perpetual problem. This issue is not solved by comprehensive two-dimensional liquid chromatography (LC × LC), but information demonstrating the success of the grafting reaction could be obtained. SEC × RPLC and HILIC × RPLC separations are presented and discussed. Full article

The activity of α-l-fucosidase (FSC) has been measured for the first time in Fiore Sardo PDO (Protected Designation of Origin) raw milk cheese. To do this, a RP-HPLC method has been developed, validated and tested on a reliable sampling of cheese experimentally produced in laboratory batches. Three experimental factors have been considered in this work: the thermal treatment undergone by the milk, the lactation period and the ripening time of cheese. Results obtained have evidenced: (i) a meaningful reduction in the activity of FSC from cheeses produced using raw milk to those obtained by thermized milk; (ii) an increase in the activity of FSC during the first months of lactation period (from December to February), followed by a substantial constancy in the central and final months of lactation (from February to May); (iii) the enzyme activity is independent of the ripening time. This method might be useful in revealing frauds related to the use of mild thermal treatments of the milk when this is not allowed as for Fiore Sardo PDO cheese but also for several PDO cheeses for which the specifications establish that raw milk only must be used for their production. Full article

Depression is considered to be one of the most prevalent mental disorders in humans. Antidepressant drugs are released in large concentrations and cause adverse effects on the environment and/or human health. Fabric Phase Sorptive Extraction (FPSE), a contemporary solid sorbent-handling technique, is a quick, sensitive, and simple analytical process. This paper describes a micro-extraction FPSE procedure coupled with High-Performance Liquid-Chromatography–Photodiode Array Detection (FPSE-HPLC–DAD) for the simultaneous extraction and analysis of five antidepressants, namely citalopram, clozapine, mirtazapine, bupropion and sertraline. Three fabric media (Whatman Cellulose filter, Whatman Microfiber Glass filter and Polylactic acid disks) and two different sol–gel sorbents (polyethylene glycol (PEG 300), alongside poly(ethylene glycol)-block-poly(propylene glycol)-block-poly(ethylene glycol) (PEG-PPG-PEG 5.800)) were tested. The best FPSE device was observed to be the microfiber glass filter coated with PEG 300 sol–gel sorbent. In addition, the parameters that affect the efficiency of the process (FPSE media and sorbents, sample pH, extraction time, elution time, etc.) were optimized. The proposed methodology displays a linear range with absolute recovery values higher than 60%, RSD% of less than 13% and LOQs in the range between 1.9–10.7 μg·L⁻¹. Finally, the method was applied in hospital and urban effluents and lake water samples, but none of the analytes were detected. Full article

Polychlorinated biphenyls (PCBs) and naphthalenes (PCNs) are ubiquitous environmental contaminants with varying degrees of toxicity. There are hundreds of possible congeners with similar chemical characteristics, which make these compounds difficult to isolate in environmental samples. Historically, PCBs and PCNs were identified by using an Aroclor or Halowax mixture instead of the individual compounds, which was impractical because of limited numbers of individual standards. A retention index database was developed with all 209 PCBs and 36 PCNs to help identify these chemicals in environmental and biological matrixes. This study uses linear and Lee retention indices to identify all 209 PCBs and 36 PCNs on nine gas chromatography columns. The most toxic congeners, the 12 dioxin-like PCBs, were compared across all columns to determine which stationary phases gave the best selectivity for those compounds. Column selectivity was also examined to determine columns for confirmatory analyses and GC×GC separations. The Rxi-17SilMS demonstrated the most drastic difference in PCB selectivity and, to a lesser extent, PCNs when compared with the other eight columns and could work as a confirmatory column or as a 2nd dimension column for GC×GC separations. Full article

Ionic liquids (ILs) are a group of non-conventional salts with melting points below 100 °C. Apart from their negligible vapor pressure at room temperature, high thermal stability, and impressive solvation properties, ILs are characterized by their tunability. Given such nearly infinite combinations of cations and anions, and the easy modification of their structures, ILs with specific properties can be synthesized. These characteristics have attracted attention regarding their use as extraction phases in analytical sample preparation methods, particularly in liquid-phase extraction methods. Given the liquid nature of most common ILs, their incorporation in analytical sample preparation methods using solid sorbents requires the preparation of solid derivatives, such as polymeric ILs, or the combination of ILs with other materials to prepare solid IL-based composites. In this sense, many solid composites based on ILs have been prepared with improved features, including magnetic particles, carbonaceous materials, polymers, silica materials, and metal-organic frameworks, as additional materials forming the composites. This review aims to give an overview on the preparation and applications of IL-based composites in analytical sample preparation in the period 2017–2020, paying attention to the role of the IL material in those composites to understand the effect of the individual components in the sorbent. Full article