Separations — Open Access Journal

In reversed-phase liquid chromatography (RPLC), positively charged basic compounds yield broad and asymmetric peaks, as a result of ionic interactions with free silanols that remain on conventional silica-based columns. Diverse solutions have been proposed to mask the silanophilic activity, which is translated to an improved peak shape. In this work, the chromatographic performance of hydrophilic interaction liquid chromatography (HILIC) was evaluated as an alternative to the addition of an ionic liquid (IL) to the aqueous-organic mobile phase used with RPLC columns, for the analysis of eight β-adrenoceptor antagonists. ILs change the behavior of RPLC stationary phases owing to adsorption on their surface. Meanwhile, in HILIC, a layer of adsorbed water is formed on the stationary phase surface. The association of cationic basic compounds with the adsorbed additive ions, hydrophilic partitioning on the HILIC columns, and other interactions, give rise to complex retention mechanisms. The chromatographic behavior was examined in terms of retention, elution strength, selectivity, peak shape and resolution, using acetonitrile-water mobile phases buffered at pH 3. Both chromatographic modes, RPLC with added IL and HILIC, proved to be a viable solution to the problem of poor peak shape for basic compounds. Full article

Recently, high demand of high-throughput analyses with high sensitivity and selectivity to molecules and drugs in different classes with different physical-chemical properties—and a reduction in analysis time—is a principal milestone for novel methodologies that researchers are trying to achieve—especially when analytical procedures are applied to clinical purposes. In addition, to avoid high doses of a single drug that could cause serious side effects, multi-drug therapies are often used to treat numerous diseases. For these reasons, the demand for methods that allow the rapid analysis of mixed compounds has increased in recent years. In order to respond to these needs, new methods and instruments have been developed. However, often the complexity of a matrix can require a long time for the preparation and processing of the samples. Different problems in terms of components, types of matrices, compounds and physical-chemical complexity are encountered when considering drugs association profiles for quantitative analyses. This review addresses not only recently optimized procedures such as chromatographic separation, but also methods that have allowed us to obtain accuracy (precision and trueness), sensitivity and selectivity in quantitative analyses for cases of drug associations. Full article

The frenetic lifestyle in the developed countries has driven us to be deficient in some nutrients, which may be overcome by supplements. Microalgae, like spirulina (Arthrospira platensis) and chlorella (Chlorella ssp.) are widely used as supplements due to their high contents of macro- and micronutrients. Chlorella and spirulina can be grown naturally in a range of water bodies, showing their high adaptability to harsh environments. They are mainly produced in countries with poor water quality and sometimes inexistent water legislation, which can be a vector of micropollutant introduction into the food chain. Thus, a method for the simultaneous determination of 31 emerging contaminants commonly found as micropollutants in freshwater (pharmaceutical and personal care products, hormones, flame retardants and biocides) in two microalgae is presented. Target contaminants were extracted from the microalgae employing ultrasound-assisted matrix solid-phase dispersion followed by gas chromatography-mass spectrometry analysis. The method was validated for chlorella and spirulina with recoveries ranging from 70% to 111% at concentrations of 25 and 100 ng·g⁻¹, and good linearity in the range from 5 to 400 ng·g⁻¹ with limits of detection below 2.5 ng·g⁻¹, in both microalgae. The method validated was applied to a range of microalgae supplement foods and the results proved that the compounds studied were below limits of detection. Full article

Molecular spectroscopic detection techniques, such as Fourier transform infrared spectroscopy (FTIR), provides additional specificity for isomers where often mass spectrometry (MS) fails, due to similar fragmentation patterns. A hyphenated system of gas chromatography (GC) with FTIR via a light-pipe interface is reported in this study to explore a number of GC–FTIR analytical capabilities. Various compound classes were analyzed—aromatics, essential oils and oximes. Variation in chromatographic peak parameters due to the light-pipe was observed via sequentially-located flame ionization detection data. Unique FTIR spectra were observed for separated mixtures of essential oil isomers having similar mass spectra. Presentation of GC×FTIR allows a ‘comprehensive’-style experiment to be developed. This was used to obtain spectroscopic/separation profiles for interconverting oxime species with their individual spectra in the overlap region being displayed on a color contour plot. Partial least square regression provides multivariate quantitative analysis of co-eluting cresol isomers derived from GC–FTIR data. The model resulted in an R² of 0.99. Prediction was obtained with R² prediction value of 0.88 and RMSEP of 0.57, confirming the method’s suitability. This study explores the potential of GC–FTIR hyphenation and re-iterates its value to derive unambiguous and detailed molecular information which is complementary to MS. Full article

Coal tar is a complex mixture of organic compounds obtained from the thermal treatment of coal; it contains several different chemical classes of compounds, such as polycyclic aromatic hydrocarbons, phenols and different heterocyclic compounds including sulphur derivatives. In the present research, a target analysis was carried out for the characterisation of fourteen different classes of organic sulphur compounds in coal tar by using cryogenically-modulated (CM) comprehensive two-dimensional gas chromatography-high resolution time-of-flight mass spectrometry (GC×GC-HR ToFMS) with the support of target analyte finding, a specific software function. Furthermore, absolute quantification data were obtained by using eight pure standard sulphur compounds, and 1-fluoronaphthalene as internal standard. Several figures-of-merit of the proposed method were measured (linearity, intra-day precision, limits of detection and quantification). Finally, the overall analytical performance of CM GC×GC-HR ToFMS was evaluated, in relation to MS similarities, mass accuracies, second-dimension peak widths, peak capacity and tailing factors. The approach proved itself as being a powerful analytical platform, benefiting from the high sensitivity, selectivity and resolving power, of both the GC and MS sides. Full article

In this work, two online comprehensive two-dimensional liquid chromatography platforms, namely Hydrophilic interaction liquid chromatography × Reversed phase (HILIC × RP) and Reversed phase × Reversed Phase (RP × RP) coupled to mass spectrometry, were compared for the analysis of complex peptide samples. In the first dimension, a HILIC Amide and C18 Bioshell peptide (150 × 2.1 mm, 1.7 and 2.0 μm) columns were selected, while, in the second dimension, a short C18 (50 × 3.0 mm, 2.7 μm) Bioshell peptide column was used. Two C18 trapping columns (10 × 3.0 mm, 1.9 μm), characterized by high retention and surface area, were employed as modulation interface in both HILIC × RP and RP × RP methods. The LC × LC platforms were coupled to UV and tandem mass spectrometry detection and tested for the separation and identification of two gastro-intestinal digests of commercial microalgae formulations (Spirulina Platensis and Klamath). Their performances were evaluated in terms of peak capacity, maximum number and properties of identified phycocyanin peptides. Our results showed that the HILIC × RP approach provided the highest peak capacity values (n_c HILIC × RP: 932 vs. n_c RP × RP: 701) with an analysis time of 60 min, while the RP × RP approach was able to identify a slight higher number of phycocyanin derived peptides (HILIC × RP: 88 vs. RP × RP: 103). These results point out the flexibility and potential of HILIC × RP and RP × RP based on trapping modulation for peptide mapping approaches. Full article

This study examines the effect of bentonite and zeolite concentration (0.25–5 g/L) on the membrane fouling of a fully automated, pilot-scale membrane bioreactor (MBR) treating high-strength synthetic municipal wastewater. Reversible fouling was estimated by sludge filterability measurements and irreversible fouling was estimated by the reduction of the carbohydrate fraction of soluble microbial products (SMP_c), which are considered to be significant MBR foulants. Both minerals were added to biomass samples (during batch-mode experiments) which were obtained from the system’s aeration tank. Results showed that the optimal bentonite and zeolite concentrations were 3.5–4 g/L and 2.5–3.5 g/L, respectively. Interestingly, above these values, the addition of both minerals increased the examined fouling indices, i.e., the measured filterability times and the SMP_c concentration, implying that they might act as foulants at high concentrations. Optical microscopy images of the biomass samples showed that the addition of minerals at the optimal concentrations did not affect significantly filamentous microorganisms, since filament index (FI) was practically unaffected (~2). Finally, regarding the system’s treating performance, it was found that the pilot-scale MBR can operate successfully with high-strength synthetic municipal wastewater, since remarkable behaviour was exhibited in terms of organics (BOD₅, COD) and ammonium (NH₄⁺-N) removal (>98%). Full article

This paper deals with the characterization of turmeric and related products using the compositional fingerprints of curcuminoids (e.g., curcumin, demethoxycurcumin and bisdemethoxycurcumin) and other phenolic compounds (e.g., hydroxybenzoic and hydroxycinnamic acids and flavonoids) as the source of analytical information. Under this approach, the quantitative determination of analytes becomes unnecessary and even data from unknown components can be advantageously exploited for sample exploration and authentication. The methodology relied on sample extraction with hydro-organic solvents to recover the components of interest and further analysis of the corresponding extracts by liquid chromatography with diode array detection (HPLC-DAD). Extraction conditions were optimized focusing on the independent recovery of curcuminoids and polyphenols. Two different HPLC methods under reversed-phase mode were used to generate the chromatographic fingerprints at 420 and 280 nm for the specific monitoring of curcuminoids and polyphenols, respectively. Both extraction and separation steps were optimized under experimental design approaches to achieve the richest compositional fingerprints in terms of variety of components. The resulting data was subsequently treated chemometrically by principal component analysis (PCA) and related classification methods to achieve a better overall description of samples. Polyphenolic fingerprints were appropriate to discriminate among turmeric and mixed spices, while curcuminoid fingerprints could be useful to distinguish turmeric varieties. Full article

Over three billion cups of coffee are consumed daily, making waste coffee grounds readily available throughout the world. Containing approximately 10–15 wt% of oil, they have great potential for biodiesel production. The goal of this work was to produce high quality biodiesel from waste coffee grounds. One fresh and four different types of waste coffee grounds were collected. Oil was extracted by the Soxhlet method with n-hexane and then purified via extractive deacidification with a potassium carbonate-based deep eutectic solvent. Biodiesels were synthesized by means of alkali catalyzed transesterification at different catalyst:methanol:oil mass ratios and reaction times. Impurities present in crude biodiesels were extracted with a choline chloride-based deep eutectic solvent. All batch extraction experiments were performed at room conditions in a small scale extractor. Optimal conditions for synthesis and purification were defined in order to assure high quality of the produced biodiesel. Additionally, continuous column extraction with the choline chloride-based solvent was tested as a purification method for crude biodiesel. Stabilization time and optimal biodiesel to solvent mass ratio were determined. The potassium carbonate-based solvent efficiently reduced the total acid number of the feedstock (deacidification efficiency ranged from 86.18 to 94.15%), while the one based on choline chloride removed free glycerol and glycerides from crude biodiesels. After continuous purification, the purified biodiesel was of excellent quality with glycerol and glyceride contents below the EN 14214 limit. Full article

In this work, Chromabond^® HLB was evaluated as an extraction sorbent of a group of seven phthalic acid esters (PAEs; i.e., dipropyl phthalate, DPP, dibutyl phthalate, DBP, diisopentyl phthalate, DIPP, di-n-pentyl phthalate, DNPP, butylbenzyl phthalate, BBP, dicyclohexyl phthalate, DCHP, and di(2-ethylhexyl)phthalate, DEHP) and one adipate (di(2-ethylhexyl) adipate, DEHA) from tap and waste water prior to gas chromatography-mass spectrometry. After the optimization of the extraction conditions (200 mg of sorbent conditioned with 10 mL of acetonitrile and 2 mL of Milli-Q water, extraction of 50 mL of water at pH 6.0, vacuum drying for 20 min and elution with 10 mL of ethyl acetate), a recovery study was developed at different concentration levels in each matrix, which revealed that most of the target analytes could be recovered between 75 and 112%, with relative standard deviation values for all of them below 20%. Matrix effect was evaluated, finding that matrix-matched calibration should be developed for most analytes in both matrices. The limits of quantification (LOQs) of the method were in the 0.82–71 ng L⁻¹ range. The developed method was also applied to the extraction of the target PAEs in different water samples finding some of them, in particular, DNPP in tap water samples, and BBP and DCHP in waste water, but below the LOQs of the method. Full article

In the present work, the kinetics of the extraction process from female inflorescences of Canapa sativa subsp. sativa var. sativa were studied, on the basis of determination of the content of cannabinoids: cannabidiolic acid (CBDA), Δ9-tetrahydrocannabinolic acid (THCA), cannabidiol (CBD) and Δ9-tetrahydrocannabinol (THC), before and after decarboxylation in the oven, in order to evaluate the possible use of the hemp extract obtained in the food sector. Therefore, both conventional maceration (CM) and rapid solid-liquid dynamic extraction (RSLDE), also known as cyclically pressurized extraction (CPE), were carried out, using parts of the plant approximately of the same size. The alcoholic extracts thus obtained were analyzed by high-performance liquid chromatography (HPLC) in order to calculate the percentages of cannabinoids present in the inflorescences and thus be able to evaluate the degree of decarboxylation. Furthermore, the extracts were dried to calculate the percentage of solid material present in it, that was made mainly by cannabinoids. The amount of substance extracted from the inflorescences was about 10% (w/w), for both cases considered. Therefore, the extraction yield was the same in the two cases examined and the final qualities were almost identical. However, the extraction times were significantly different. In fact, the maceration of hemp inflorescences in ethyl alcohol was completed in no less than 24 h, while with the RSLDE the extraction was completed in only 4 h. Finally, for a better understanding of the extraction process with cyclically pressurized extraction, a numerical simulation was carried out which allowed to better evaluate the influence of extractive parameters. Full article

An ultra-performance liquid chromatography–tandem mass spectrometry (UPLC–MS/MS) method, in combination with a mixed matrix membrane microextraction method for the quantification of nonsteroidal anti-inflammatory drugs (NSAIDs) in environmental water samples, is reported. The extraction device was prepared by casting well-dispersed polymeric bonded octadecyl (C₁₈) particles in a cellulose triacetate matrix solution onto commercially available 200 μL micropipette tips. The membrane formed contains 25% of the adsorbent loading amount and was firmly attached to the inner wall of the membrane tip. The dynamic extraction was performed by withdrawing and dispensing the sample solution through the tip device for effective analyte adsorption, followed by the analyte desorption process into 40 μL of methanol and acetonitrile (1:1) prior to UPLC–MS/MS analysis. NSAIDs—namely diclofenac, ibuprofen, indoprofen, naproxen and sulindac—were chosen as targeted analytes. Several extraction parameters were comprehensively optimized, including sample pH value, ionic strength, dynamic extraction cycle, desorption solvent and desorption time. The optimized conditions demonstrated a linear range from 0.25 to 500 ng L⁻¹, with correlation coefficients (r²) from 0.9988 to 0.9992 and detection limits ranging from 0.08 to 0.40 ng L⁻¹. The recoveries of the spiked water samples were between 92% and 99% and exhibited excellent precision relative to standard deviations (RSDs ≤ 4.9%), and enrichment factors (EFs) were at 201–249 for the developed approach. Full article

In this study, a metabolomic approach was used to investigate the effect of seasonality on the chemical composition and yield of anti-inflammatory active principle, α-humulene, in the essential oil of three genotypes of Varronia curassavica Jacq. (Syn. Cordia verbenaceae). The essential oils were extracted by hydrodistillation and analyzed by comprehensive two-dimensional gas chromatography coupled to mass spectrometry (GC×GC-MS). The GC×GC approach a three-fold improvement in qualitative analysis (48 compounds were identified by GC-MS versus 135 by GC×GC-MS). The improved resolving power of GC×GC resolved important coelutions and enabled the detection of unusual substances in V. curassavica essential oil. The chromatographic data was analyzed by using peak table-based chemometrics, namely, principal component analysis (PCA) and hierarchical cluster analysis (HCA). The metabolic study showed that seasonality has a significant effect on the chemical composition. The α-humulene content was affected by genotype and season. Spring and summer were the best harvest seasons for the yield of the active ingredient, found in higher concentrations in the VC2 genotype. The proposed metabolomic workflow was successfully applied to terpene analysis found in V. curassavica essential oil, and such results have broadened our understanding of the influence of seasonal factors on the specialized metabolism of the species. Full article

In this research paper, we report a hyphenated technique based on ultra-high performance liquid chromatography–tandem mass spectrometry for the determination of twelve Perfluorinated Alkylated Substances in surface and groundwater samples from Lombardia Region during the monitoring activities in 2018 as new emerging and toxic pollutants. A green analytic method, developed by using an online Solid Phase Extraction coupled with UHPLC-MS/MS and previously validated, was applied for 4992 determinations conducted on 416 samples from 109 different sampling stations. Among the results, PFOS, PFOA, PFBA, PFBS, PFPeA and PFHxA were identified as the most abundant analytes detected. PFASs concentrations, in most cases, were below the limits of quantification and, in the cases where the limits of quantification have been exceeded, the values found were lower than Italy directive. PFOS is an exception and in fact this compound was detected in 76% of analyzed samples (surface and ground waters). Solid phase extraction with high performance liquid chromatography–tandem Mass Spectrometry has proved to be a very good Hyphenated techniques able to detect low concentrations of pollutants in surface and groundwater samples. Full article

In the present study, we report the results of our investigation of the oxidative forced degradation of glutathione in its nutraceutical formulations by two validated analytical methods. The first is based on the reaction of glutathione with o-phthalaldehyde through an automated zone fluidics flow platform and fluorimetric detection (λ_ex/λ_em = 340/425 nm). The second is based on the separation of glutathione and its oxidation product by a green reversed-phase HPLC method coupled to direct UV detection, at 210 nm. A solution of 3% w/v H₂O₂ provided fast oxidation of more than 95% of glutathione to yield oxidized glutathione in a time period of 180 min. The mechanism of the oxidation was proved to follow pseudo-first order kinetics. The k, t₉₀ and t_1/2 values were calculated. Full article

Pesticides and veterinary pharmaceuticals are used for effective crop production and prevention of livestock diseases; these chemicals are released into the environment via various pathways. Although the chemicals are typically present in trace amounts post-release, they could disturb aquatic ecosystems and public health through resistance development toward drugs or diseases, e.g., reproductive disorders. Thus, the residues of pesticides and veterinary pharmaceuticals in the environment must be managed and monitored. To that end, we developed a simultaneous analysis method for 41 target chemicals in environmental water samples using ultra-high-performance liquid chromatography (UHPLC)–quadrupole-orbitrap high-resolution mass spectrometry (HRMS) coupled with an on-line solid-phase extraction system. Calibration curves for determining linearity were constructed for 10–750 ng∙L⁻¹, and the coefficient of determination for each chemical exceeded 0.99. The method’s detection and quantitation limits were 0.32–1.72 ng∙L⁻¹ and 1.02–5.47 ng∙L⁻¹, respectively. The on-line solid-phase extraction system exhibited excellent method reproducibility and reduced experimental error. As the proposed method is applicable to the monitoring of pesticides and veterinary pharmaceuticals in surface water and groundwater samples acquired near agricultural areas, it allows for the management of chemicals released into the environment. Full article

Historically, one-dimensional gas chromatography combined with mass spectrometry (GC/MS) has been employed in the analysis of petrochemical samples such as diesel, crude oil and bitumen. With increasingly complex samples, obtaining detailed information can be difficult with this method due to the large number of coelutions. By implementing comprehensive two-dimensional gas chromatography time-of-flight mass spectrometry (GC×GC-TOFMS), the limitations of GC/MS can be overcome, due to the ability of this method to separate mixtures using two different separation mechanisms and obtain full mass spectra. Furthermore, this enables an investigation of biomarkers, compounds which aid in the identification of geological and environmental processes, potentially differentiating crude oil samples. Cryogenic-based thermal modulators are typically used for this application due to their superior focusing effect; however, some platforms require expensive cryogenic consumables. The solid-state modulator (SSM), a cryogen-free thermal platform, was employed for the first time for the group and biomarker analysis of Alberta oil sands bitumen. Evaluation of the SSM performance was based on published literature data on bitumen analysis. Extracted ion chromatograms (EIC) and molecular ion peaks were used for the confirmation of the groups’ and individual’s analytes. Identification of the characteristic biomarkers responsible for determining thermal maturity, source rock or oil origin was achieved. These results indicate the successful analysis of bitumen by consumable-free, solid-state modulation-based GC×GC-TOFMS. Full article

Thirty years since the invention and public disclosure of solid phase microextraction (SPME), the technology continues evolving and inspiring several other green extraction technologies amenable for the collection of small molecules present in complex matrices. In this manuscript, we review the fundamental and operational aspects of a novel SPME geometry that can be used to “hunt” target molecules in complex matrices: the SPME Arrow. In addition, a series of applications in environmental, food, cannabis and forensic analysis are succinctly covered. Finally, special emphasis is placed on novel interfaces to analytical instrumentation, as well as recent developments in coating materials for the SPME Arrow. Full article