Separations, Volume 6, Issue 1 (March 2019) – 18 articles

A method for the analysis of thyroid hormones by liquid chromatography-mass spectrometry was used for the dissolution testing of single- and dual-component thyroid hormone supplements via a two-stage biorelevant dissolution procedure. The biorelevant media consisted of fasted-state simulated gastric fluid and fasted state simulated intestinal fluid at 37 °C, and was investigated using an internationally recognized protocol. The dissolution profiles showed consistent solubilization for both single- and dual-component batches at pH 6.5 in the fasted-state simulated intestinal fluid. Full article

Per and polyfluoroalkyl substances (PFASs) are a huge class of Contaminants of Emerging Concern, well-known to be persistent, bioaccumulative and toxic. They have been detected in different environmental matrices, in wildlife and even in humans, with drinking water being considered as the main exposure route. Therefore, the present study focused on the estimation of PFAS in the Metropolitan Area of Turin, where SMAT (Società Metropolitana Acque Torino S.p.A.) is in charge of the management of the water cycle and the development of a tool for supporting “smart” water quality monitoring programs to address emerging pollutants’ assessments using multivariate spatial and statistical analysis tools. A new “green” analytical method was developed and validated in order to determine 16 different PFAS in drinking water with a direct injection to the Ultra High Performance Liquid Chromatography tandem Mass Spectrometry (UHPLC-MS/MS) system and without any pretreatment step. The validation of this analytical method resulted in really low Quantification Limits (5 ng L⁻¹), in satisfying recoveries (70%–102%) and in a good linearity (R² = 0.99) for every compound. Among the results, only 4 compounds and only 6% of the samples showed a pollution level higher than the limits of and Quantification (LOQ). Finally, the correlation study between the assessment findings and the industrial sites which serve as potential sources of pollution in the monitored area was carried out. Full article

A method involving the collection and determination of organic and inorganic gunshot residues on hands using on-line in-tube solid-phase microextraction (IT-SPME) coupled to miniaturized capillary liquid chromatography with diode array detection (CapLC-DAD) and scanning electron microscopy coupled to energy dispersion X-ray (SEM-EDX), respectively, for quantifying both residues was developed. The best extraction efficiency for diphenylamine (DPA) as the main target among organic residues was achieved by using a dry cotton swab followed by vortex-assisted extraction with water, which permits preservation of inorganic residues. Factors such as the nature and length of the IT-SPME extractive phase and volume of the sample processed were investigated and optimized to achieve high sensitivity: 90 cm of TRB-35 (35% diphenyl, 65% polydimethylsiloxane) capillary column and 1.8 mL of the processed sample were selected for the IT-SPME. Satisfactory limit of detection of the method for analysis of DPA deposited on shooters’ hands (0.3 ng) and precision (intra-day relative standard deviation, 9%) were obtained. The utility of the described approach was tested by analyzing several samples of shooters’ hands. Diphenylamine was found in 81% of the samples analyzed. Inorganic gunshot residues analyzed by SEM-EDX were also studied in cotton swab and lift tape kit samplers. Optical microscopy was used to see the inorganic gunshot residues in the cotton swab samplers. The lift tape kits provided lesser sensitivity for DPA than dry cotton swabs—around fourteen times. The possibility of environmental and occupational sources could be eliminated when DPA was found together with inorganic residues. Then, the presence of inorganic and organic residues in a given sample could be used as evidence in judicial proceedings in the forensic field. Full article

Line-start incremental centrifugal liquid sedimentation (disc-CLS) is a powerful technique to determine particle size based on the principles of Stokes’ law. As most input quantities of the Stokes equation cannot be easily determined for typical instruments used for this method, an alternative method which depends on calibrating the sedimentation time scale with reference particles has become common practice. Unfortunately, most of these calibration materials (calibrants) come with limited information regarding their metrological reliability (e.g., lack of measurement uncertainties and traceability statements, incomplete measurand definitions). As a consequence, routine particle size results obtained by disc-CLS are mostly only traceable to the calibrant used, and effective comparisons can only be made for those results originating from measurements performed with the same types of calibrants. In this study, we discuss the concept of metrological traceability and demonstrate that particle size results obtained by disc-CLS can be traceable to the ultimate metrological reference, i.e., the unit of length in the International System of Units (SI), the meter. Using the example of two colloidal silica certified reference materials, we describe how laboratories can realize metrological traceability to the SI by simplifying complex traceability networks. Full article

Dairy fat is one of the most complex natural fats because of its fatty acid (FA) composition. Ruminant dairy fat contains more than 400 different FA varying in carbon chain length, and degree, position and configuration of unsaturation. The following article reviews the different methods available to analyze FA (both total and free) in milk and dairy products. The most widely used methodology for separating and analyzing dairy FA is gas chromatography, coupled to a flame ionization detector (CG-FID). Alternatively, gas chromatography coupled to a mass spectrometer (GC-MS) is also used. After lipid extraction, total FA (TFA) are commonly converted into their methyl esters (fatty acid methyl esters, FAME) prior to chromatographic analysis. In contrast, free FA (FFA) can be analyzed after conversion to FAME or directly as FFA after extraction from the product. One of the key questions when analyzing FAME from TFA is the selection of a proper column for separating them, which depends mainly on the objective of the analysis. Quantification is best achieved by the internal standard method. Recently, near-infrared spectroscopy (NIRS), Raman spectroscopy (RS) and nuclear magnetic resonance (NMR) have been reported as promising techniques to analyze FA in milk and dairy products. Full article

In-tube solid-phase microextraction (IT-SPME) coupled on-line to miniaturized liquid chromatography (LC) has emerged as a powerful tool to address a variety of analytical problems. However, in order to expand its applicability, the development of new sorbents that enhance the efficiency and specificity of the extraction is highly desirable. In this respect, the employment of capillary columns coated with sorbents functionalized with nanoparticles (NPs) replacing the loop of the injection valve (in-valve IT-SPME) is one of the most attractive options. In this work, polymers of tetraethyl orthosilicate (TEOS) and trimethoxyethylsilane (MTEOS) modified with SiO₂ and TiO₂ NPs have been synthetized and used for the extraction of a variety of water pollutants, using both Capillary-LC and Nano-LC. Compounds with different chemical structures and polarities such as the artificial sweetener saccharine, the polycyclic aromatic hydrocarbons (PAHs) naphthalene and fluoranthene, and some phenylurea and organophosphorous herbicides have been used as target analytes. The extraction efficiencies found with the synthetized capillaries have been compared to those obtained with commercially available capillaries coated with polydiphenyl-polydimethylsiloxane (PDMS), nitroterephthalic acid modified polyetilenglicol (FFAP), and polystyrene-divinylbenzene (PS-DVB) phases. The results obtained in this preliminary study showed that, although PS-DVB phase has the strongest affinity for compounds with two or more aromatic rings, the extraction with TEOS-MTEOS coatings modified with NPs is the best option for a majority of the tested compounds. Examples of application are given. Full article

This contribution aimed to measure for the first time the amount of biogenic amines (BAs) in one of the most ancient and traditional sheep cheese produced in Sardinia, Italy: the Protected Designation of Origin (PDO) Fiore Sardo. To achieve this, an original RP-HPLC-DAD-UV method has been developed that was completely validated in terms of LoD, LoQ, linearity, precision and trueness, and tested on 36 real Fiore Sardo PDO cheese samples produced by four different cheesemakers and marketed by four stores. The average total concentration of the eight BAs (i.e., tyramine, tryptamine, histidine, putrescine, cadaverine, 2-phenylethylamine, spermine and spermidine) measured in Fiore Sardo cheese was 700 mg/kg, with a range between 170 mg/kg and 1100 mg/kg. A great variability in the total amount of BAs has been evidenced among the Fiore Sardo marketed in the four stores as well as for the cheeses purchased in different times in the same store. Tyramine (350 mg/kg), putrescine (150 mg/kg), histamine (80 mg/kg) and cadaverine (30 mg/kg) are the most abundant BAs found in this matrix. Among the many factors concurring, the dominant microflora of Fiore Sardo PDO is likely the principal cause of the qualitative and quantitative distribution of BAs in this matrix. Finally, the total amount of BAs found in Fiore Sardo PDO is not able to cause any health alert situation for consumers. Full article

The hydroalcoholic extract of leaves and twigs of Lindera neesiana (Wall. ex Nees) Kurz (Lauraceae) was subjected to various column chromatographic methods that isolated five kaempferol glycosides: kaempferol 3-O-β-glucopyranosyl(1→2)-[α-rhamnopyranosyl-(1→6)]-β- glucopyranoside-7-O-α-rhamnopyranoside (1); kaempferol 3-O-β-glucopyranosyl-(1→2)-[α-rhamnopyranosyl (1→6)]-β-glucopyranoside (2); kaempferol 3-O-β-glucopyranosyl(1→2)- α-rhamnopyranoside-7-O-α-rhamnopyranoside (3); kaempferol 3-O-sophoroside (4); and kaempferol 3-O-α-rhamnopyranoside (5). The extract showed moderate free radical scavenging activity and potent pancreatic lipase inhibitory activity. Full article

In the over 1,800 articles published since their inception in 2001, most deep eutectic solvents (DES) synthesized have been hydrophilic. The low cost, low toxicity, and bioavailability of DES make the solvent ‘green’ and sustainable for diverse applications. Conversely, the hydrophilicity of DES limits their practical application to only polar compounds, which is a major drawback of the solvent. For the past three years, hydrophobic deep eutectic solvents (HDES) have emerged as an alternative extractive media capable of extracting non-polar organic and inorganic molecules from aqueous environments. Due to the infancy of HDES, for the first time, this mini-review summarizes the recent developmental advances in HDES synthesis, applications, challenges, and future perspectives of the solvent. In the future, it is believed HDES will replace the majority of toxic organic solvents used for analytical purposes. Full article

The seeds of the Gac fruit, Momordica cochinchinensis Spreng, are rich in trypsin inhibitors (TIs) but their optimal extraction and the effects of freeze drying are not established. This study aims to (1) compare aqueous solvents (DI water, 0.1 M NaCl, 0.02 M NaOH and ACN)/water/FA, 25:24:1) for extracting TIs from defatted Gac seed kernel powder, (2) to optimise the extraction in terms of solvent, time and material to solvent ratio and (3) to produce a TI-enriched freeze-dried powder (FD-TIP) with good characteristics. Based on the specific TI activity (TIA), the optimal extraction was 1 h using a ratio of 2.0 g of defatted powder in 30 mL of 0.05 M NaCl. The optimisation improved the TIA and specific TIA by 8% and 13%, respectively. The FD-TIP had a high specific TIA (1.57 ± 0.17 mg trypsin/mg protein), although it also contained saponins (43.6 ± 2.3 mg AE/g) and phenolics (10.5 ± 0.3 mg GAE/g). The FD-TIP was likely stable during storage due to its very low moisture content (0.43 ± 0.08%) and water activity (0.18 ± 0.07) and its ability to be easily reconstituted in water due to its high solubility index (92.4 ± 1.5%). Therefore, the optimal conditions for the extraction of TIs from defatted Gac seed kernel powder followed by freeze drying gave a high quality powder in terms of its highly specific TIA and physical properties. Full article

The gas chromatographic retention behaviour of 16 polycyclic aromatic hydrocarbons (PAHs) and alkylated PAHs on a new ionic liquid stationary phase, 1,12-di(tripropylphosphonium) dodecane bis(trifluoromethanesulfonyl)imide (SLB^®-ILPAH) intended for the separation of PAH mixtures, was compared with the elution pattern on more traditional stationary phases: a non-polar phenyl arylene (DB-5ms) and a semi-polar 50% phenyl dimethyl siloxane (SLB PAHms) column. All columns were tested by injections of working solutions containing 20 parental PAHs from molecular weight of 128 to 278 g/mol and 48 alkylated PAHs from molecular weight of 142 to 280 g/mol on a one dimensional gas chromatography-mass spectrometry (GC-MS) system. The SLB PAHms column allowed separation of most isomers. The SLB^®-ILPAH column showed a rather different retention pattern compared to the other two columns and, therefore, provided a potential for use in comprehensive two-dimensional GC (GC×GC). The ionic liquid column and the 50% phenyl column showed good thermal stability with a low bleed profile, even lower than that of the phenyl arylene “low bleed” column. Full article

Bisphosphonates are used to treat various skeletal disorders, as they modulate bone metabolism by inhibition of the osteoclast-mediated bone resorption. These compounds are both polar and ionic, and therefore, by using reversed phase liquid chromatography are eluted rapidly. Hydrophilic interaction liquid chromatography (HILIC) is an advantageous technique for the separation and analysis of polar molecules. As the elution order in HILIC is reversed to reversed phase liquid chromatography, a reasonable retention and selectivity for polar compounds is expected. In this work the retention mechanism of three bisphosponates, namely risedronate, tiludronate and zoledronate, was investigated under zwitterionic HILIC conditions. The key factors influencing the retention of the analytes on a zwitterionic ZIC®-pHILIC column (150.0 × 2.1 mm i.d., 200 Å, 3.5 μm) have been systematically investigated. It was found that apart from partition, electrostatic repulsions play an important role in the retention of bisphosphonates. Peak tailing of risedronate and zoledronate was improved by the addition of sodium pyrophosphate in the mobile phase. A zwitterionic hydrophilic interaction liquid chromatography-photodiode array (HILIC-PDA) method was further optimized and fully validated to quantitate risedronate in commercial film-coated tablets. The calibration curves for risedronate showed good linearity (r > 0.9991) within the calibration range tested. The intra- and inter-day coefficient of variation (CV) values was less than 0.6%, while the relative percentage error (%Er) was less than −2.3%. Accelerated stability studies of risedronate conducted under several degradation conditions including hydrolysis, oxidation and heat demonstrated the selectivity of the procedure. A short-run analysis of not more than 6 min allowed the analysis of large samples per day. The applicability of the method for the quantitation of risedronate was demonstrated via the analysis of commercial tablets containing this compound. Full article

Mass spectrometry (MS)-based precursor ion isolation, collision-induced dissociation (CID) fragmentation, and detection using linear ion-trap multiple-stage mass spectrometry (LIT MSⁿ) in combination with high resolution mass spectrometry (HRMS) provides a unique tool for structural characterization of complex mixture without chromatographic separation. This approach permits not only separation of various lipid families and their subfamilies, but also stereoisomers, thereby, revealing the structural details. In this report, we describe the LIT MSⁿ approach to unveil the structures of a 2,3-diacyl trehalose (DAT) family isolated from the cell envelope of Mycobacterium tuberculosis, in which more than 30 molecular species, and each species consisting of up to six isomeric structures were found. LIT MSⁿ performed on both [M + Na]⁺ and [M + HCO₂]⁻ ions of DAT yield complimentary structural information for near complete characterization of the molecules, including the location of the fatty acyl substituents on the trehalose backbone. This latter information is based on the findings of the differential losses of the two fatty acyl chains in the MS² and MS³ spectra; while the product ion spectra from higher stage LIT MSⁿ permit confirmation of the structural assignment. Full article

Gold nanoparticles (AuNPs) are increasingly studied for cancer treatment purposes, as they can potentially improve both control and efficiency of the treatment. Intensive research is conducted in vitro on rodent and human cell lines to objectify the gain of combining AuNPs with cancer treatment and to understand their mechanisms of action. However, using nanoparticles in such studies requires thorough knowledge of their cellular uptake. In this study, we optimized single particle ICPMS (sp-ICPMS) analysis to qualify and quantify intracellular AuNP content after exposure of in vitro human breast cancer cell lines. To this aim, cells were treated with an alkaline digestion method with 5% TMAH, allowing the detection of gold with a yield of 97% on average. Results showed that under our experimental conditions, the AuNP size distribution appeared to be unchanged after internalization and that the uptake of particles depended on the cell line and on the exposure duration. Finally, the comparison of the particle numbers per cell with the estimates based on the gold masses showed excellent agreement, confirming the validity of the sp-ICPMS particle measurements in such complex samples. Full article

The high performance of chemically-modified silica gel packing materials is based on the utilization of pure silica gels. Earlier silica gels used to be made from inorganic silica; however, nowadays, silica gels are made from organic silanes. The surface smoothness and lack of trace metals of new silica gels permits easy surface modifications (chemical reactions) and improves the reproducibility and stability. Sharpening peak symmetry is based on developing better surface modification methods (silylation). Typical examples can be found in the chromatography of amitriptyline for silanol testing and that of quinizarin for trace metal testing. These test compounds were selected and demonstrated sensitive results in the measurement of trace amounts of either silanol or trace metals. Here, we demonstrate the three-dimensional model chemical structures of bonded-phase silica gels with surface electron density for easy understanding of the molecular interaction sites with analytes. Furthermore, a quantitative explanation of hydrophilic and hydrophobic liquid chromatographies was provided. The synthesis methods of superficially porous silica gels and their modified products were introduced. Full article

Antibody-drug conjugates (ADCs) are promising state-of-the-art biopharmaceutical drugs for selective drug-delivery applications and the treatment of diseases such as cancer. The idea behind the ADC technology is remarkable as it combines the highly selective targeting capacity of monoclonal antibodies with the cancer-killing ability of potent cytotoxic agents. The continuous development of improved ADCs requires systematic studies on the nature and effects of warhead modification. Recently, we focused on the hydrophilic modification of monomethyl auristatin E (MMAE), the most widely used cytotoxic agent in current clinical trial ADCs. Herein, we report on the use of micellar electrokinetic chromatography (MEKC) for studying the hydrophobic character of modified MMAE derivatives. Our data reveal a connection between the hydrophobicity of the modified warheads as free molecules and their cytotoxic activity. In addition, MMAE-trastuzumab ADCs were constructed and evaluated in preliminary cytotoxic assays. Full article