Dependency of fentanyl analogue protomer ratios on solvent conditions as measured by ion mobility-mass spectrometry.

Recently, our group has shown that fentanyl and many of its analogues form prototropic isomers ("protomers") during electrospray ionization. These different protomers can be resolved using ion mobility spectrometry and annotated using mobility-aligned tandem mass spectrometry fragmentation. However, their formation and the extent to which experimental variables contribute to their relative ratio remain poorly understood. In the present study, we systematically investigated the effects of mixtures of common chromatographic solvents (water, methanol, and acetonitrile) and pH on the ratio of previously observed protomers for 23 fentanyl analogues. Interestingly, these ratios (N-piperidine protonation vs. secondary amine/O = protonation) decreased significantly for many analogues (e.g., despropionyl ortho-, meta-, and para-methyl fentanyl), increased significantly for others (e.g., cis-isofentanyl), and remained relatively constant for the others as solvent conditions changed from 100% organic solvent (methanol or acetonitrile) to 100% water. Interestingly, pH also had significant effects on this ratio, causing the change in ratio to switch in many cases. Lastly, increasing conditions to pH ≥ 4.0 also prompted the appearance of new mobility peaks for ortho- and para-methyl acetyl fentanyl, where all previous studies had only showed one single distribution. Because these ratios have promise to be used qualitatively for identification of these (and emerging) fentanyl analogues, understanding how various conditions (i.e., mobile phase selection and/or chromatographic gradient) affect their ratios is critically important to the development of advanced ion mobility and mass spectrometry methodologies to identify fentanyl analogues.

Facile Access to Solifenacin Impurity K: One-Step Synthesis and an HPLC-MS Method for Its Determination.

Solifenacin (SFC) is a potent muscarinic antagonist that effectively reduces bladder muscle contraction, thereby alleviating symptoms such as frequency of micturition and urgency. Oxidation of SFC leads to the formation of impurities like Impurity K. Effective analysis and control of this impurity is crucial for ensuring compliance with regulatory standards and safeguarding patient health. To address these challenges, we propose a novel one-step synthesis of Impurity K from SFC. Impurity K was synthesized using cerium(IV) ammonium nitrate (CAN) in water/acetonitrile as the solvent. Additionally, we describe a new HPLC-MS method for the detection of Impurity K in solifenacin succinate tablets.

Real samples sensitive dopamine sensor based on poly 1,3-benzothiazol-2-yl((4-carboxlicphenyl)hydrazono)acetonitrile on a glassy carbon electrode.

Herein, a novel electrochemical sensor that was used for the first time for sensitive and selective detection of dopamine (DA) was fabricated. The new sensor is based on the decoration of the glassy carbon electrode surface (GC) with a polymer film of 1,3-Benzothiazol-2-yl((4-carboxlicphenyl)hydrazono)) acetonitrile (poly(BTCA). The prepared (poly(BTCA) was examined by using different techniques such as 1H NMR, 13C NMR, FTIR, and UV-visible spectroscopy. The electrochemical investigations of DA were assessed using cyclic voltammetry (CV) and differential pulse voltammetry (DPV). The results obtained showed that the modifier increased the electrocatalytic efficiency with a noticeable increase in the oxidation peak current of DA in 0.1 M phosphate buffer solution (PBS) at an optimum pH of 7.0 and scan rate of 200 mV/s when compared to unmodified GC. The new sensor displays a good performance for detecting DA with a limit of detection (LOD 3σ), and limit of quantification (LOQ 10σ) are 0.28 nM and 94 nM respectively. The peak current of DA is linearly proportional to the concentration in the range from 0.1 to 10.0 µM. Additionally, the fabricated electrode showed sufficient reproducibility, stability, and selectivity for DA detection in the presence of different interferents. The proposed poly(BTCA)/GCE sensor was effectively applied to detect DA in the biological samples.

Effects of Fe(III) on the formation and toxicity alteration of halonitromethanes, dichloroacetonitrile, and dichloroacetamide from polyethyleneimine during UV/chlorine disinfection.

Trace iron ions (Fe(III)) are commonly found in water and wastewater, where free chlorine is very likely to coexist with Fe(III) affecting the disinfectant's stability and N-DBPs' fate during UV/chlorine disinfection, and yet current understanding of these mechanisms is limited. This study investigates the effects of Fe(III) on the formation and toxicity alteration of halonitromethanes (HNMs), dichloroacetonitrile (DCAN), and dichloroacetamide (DCAcAm) from polyethyleneimine (PEI) during UV/chlorine disinfection. Results reveal that the maxima concentrations of HNMs, DCAN, and DCAcAm during UV/chlorine disinfection with additional Fe(III) were 1.39, 1.38, and 1.29 times higher than those without additional Fe(III), instead of being similar to those of Fe(III) inhibited the formation of HNMs, DCAN and DCAcAm during chlorination disinfection. Meanwhile, higher Fe(III) concentration, acidic pH, and higher chlorine dose were more favorable for forming HNMs, DCAN, and DCAcAm during UV/chlorine disinfection, which were highly dependent on the involvement of HO· and Cl·. Fe(III) in the aquatic environment partially hydrolyzed to the photoactive Fe(III)­hydroxyl complexes Fe(OH)2+ and [Fe(H2O)6]3+, which undergone UV photoactivation and coupling reactions with HOCl to achieve effective Fe(III)/Fe(II) interconversion, a process that facilitated the sustainable production of HO·. Extensive product analysis and comparison verified that the HO· production enhanced by the Fe(III)/Fe(II) internal cycle played a primary role in increasing HNMs, DCAN, and DCAcAm productions during UV/chlorine disinfection. Note that the incorporation of Fe(III) increased the cytotoxicity and genotoxicity of HNMs, DCAN, and DCAcAm formed during UV/chlorine disinfection, and yet Fe(III) did not have a significant effect on the acute toxicity of water samples before, during, and after UV/chlorine disinfection. The new findings broaden the knowledge of Fe(III) affecting HNMs, DCAN, and DCAcAm formation and toxicity alteration during UV/chlorine disinfection.

Exploring the solubility and intermolecular interactions of biologically significant amino acids l-serine and L-cysteine in binary mixtures of H2O + DMF, H2O + DMSO and H2O + ACN in temperature range from T = 288.15 K to 308.15 K.

In the presented work, a study on the solubility and intermolecular interactions of l-serine and L-cysteine was carried out in binary mixtures of H2O + dimethylformamide (DMF), H2O + dimethylsulfoxide (DMSO), and H2O + acetonitrile (ACN) in the temperature range of T = 288.15 K to 308.15 K. l-serine exhibited the highest solubility in water, while L-cysteine was more soluble in water-DMF. The solvation process was assessed through standard Gibbs energy calculations, indicating the solvation stability order: water-ACN > water-DMSO > water-DMF for l-serine, and water-DMF > water-DMSO > water-ACN for L-cysteine. This study also explored the influence of these amino acids on solvent-solvent interactions, revealing changes in chemical entropies and self-association patterns within the binary solvent mixtures.

Guidelines for descriptor assignments for the solvation parameter model by separation techniques.

The solvation parameter model uses six compound descriptors to model equilibrium properties in biphasic systems formally defined as excess molar refraction, E, dipolarity/polarizability, S, overall hydrogen-bond acidity, A, overall hydrogen-bond basicity, B, McGowan's characteristic volume, V, and the gas-liquid partition constant on hexadecane at 25 °C, L. The V descriptor can be assigned from structure and the E descriptor for compounds liquid at 20 °C can be calculated from its refractive index and characteristic volume. The E descriptor for compounds solid at 20 °C and the S, A, B, and L descriptors are assigned from experimental properties traditionally obtained by chromatographic, liquid-liquid partition, and solubility measurements. Here I report an efficient experimental design using the Solver method for the accurate assignment of descriptors for neutral compounds that simultaneously minimizes laboratory resources. This multi-technique approach requires 3 retention factor measurements in a 60 °C temperature range per compound on four columns by gas chromatography, 3 retention factor measurements in a 30 % (v/v) acetonitrile composition range per compound on two columns by reversed-phase liquid chromatography, and eight partition constant measurements by liquid-liquid partition in totally organic and aqueous biphasic systems for a total of 26 experimental measurements. The accuracy of the descriptor assignments was validated by comparison with the values in the Wayne State University (WSU) descriptor database taken as the best estimate of the true descriptor values. The E, S, A, B and L descriptors were assigned simultaneously by the Solver method using the above approach without significant bias and with an average absolute deviation (AAD) of 0.054, 0.018, 0.015, 0.013, and 0.040, respectively, compared with the WSU database values, corresponding to a relative absolute average deviation in percent (RAAD) of 7.2, 1.9, 3.6, 5.1, and 0.84 %, respectively, for 32 varied compounds. This streamlined approach represents a significant improvement on earlier single-technique approaches used as the starting point for the development of the multi-technique approach. For compounds of variable hydrogen-bond basicity modifications to the multi-technique approach were implemented while maintaining the same number of experimental measurements. Acceptable descriptor assignments for B/B° were obtained for compounds liquid at 20 °C for which the E descriptor was available by calculation. For solid compounds at 20 °C the E and B/B° descriptors are restricted to qualitative application where approximate values may be acceptable.

Organic-solvent ditch overlap in reversed-phase liquid chromatography: A molecular dynamics simulation study in cylindrical 6-12 nm-diameter pores.

Mass transport through the mesopore space of a reversed-phase liquid chromatography (RPLC) column depends on the properties of the chromatographic interface, particularly on the extent of the organic-solvent ditch that favors the analyte surface diffusivity. Through molecular dynamics simulations in cylindrical RPLC mesopore models with pore diameters between 6 and 12 nm we systematically trace the evolution of organic-solvent ditch overlap due to spatial confinement in the mesopore space of RPLC columns for small-molecule separations. Each pore model of a silica-based, endcapped, C18-stationary phase is equilibrated with two mobile phases of comparable elution strength, namely 70/30 (v/v) water/acetonitrile and 60/40 (v/v) water/methanol, to consider the influence of the mobile-phase composition on the onset of organic-solvent ditch overlap. The simulations show that, as the pore diameter decreases from 9 to 6 nm, the bonded-phase density extends and compacts towards the pore center, which leads to increased accumulation of organic-solvent excess and thus enhanced organic-solvent diffusivity in the ditch. Because the acetonitrile ditch is more pronounced than the methanol ditch, acetonitrile ditch overlap sets in at less severe spatial confinement than methanol ditch overlap. The pore-averaged methanol and acetonitrile diffusivities are considerably raised by ditch overlap in the 6 nm-diameter pore, but also benefit from the ditch (without overlap) in the 7 to 12 nm-diameter pores, whereby local and pore-averaged effects are generally larger for acetonitrile than methanol.

Time-resolved keto-enol tautomerization of the medicinal pigment curcumin.

Curcumin is a medicinal agent that exhibits anti-cancer and anti-Alzheimer's disease properties. It has a keto-enol moiety that gives rise to many of its chemical properties including metal complexation and acid-base equilibria. A previous study has shown that keto-enol tautomerization at this moiety is implicated in the anti-Alzheimer's disease effect of curcumin, highlighting the importance of this process. In this study, tautomerization of curcumin in methanol, acetone and acetonitrile was investigated using time-resolved 1H nuclear magnetic resonance spectroscopy. Curcumin undergoes hydrogen-deuterium exchange with the solvents and the proton resonance peak corresponding to the hydrogen at the α-carbon position (Cα) decays as a function of time, signifying deuteration at this position. Because tautomerization is the rate limiting step in the deuteration of curcumin at the Cα position, the rate of tautomerization is inferred from the rate of deuteration. The rate constant of tautomerization of curcumin shows a temperature dependence and analysis using the Arrhenius equation revealed activation energies (Ea) of tautomerization of (80.1 ± 5.9), (64.1 ± 1.0) and (68.3 ± 5.5) kJ mol-1 in methanol, D2O/acetone and D2O/acetonitrile, respectively. Insight into the role of water in tautomerization of curcumin was further offered by density functional theory studies. The transition state of tautomerization was optimized in the presence of water molecules. The results show a hydrogen-bonded solvent bridge between the diketo moiety and Cα of curcumin. The Ea of tautomerization of curcumin shows a strong dependence on the number of water molecules in the solvent bridge, indicating the critical role played by the solvent bridge in catalyzing tautomerization of curcumin.

Preparation and characterization of stationary phase gradients on C8 liquid chromatography columns.

Continuous C8 stationary phase gradients are created on commercial Waters Symmetry Shield RP8 columns by strategically cleaving the C8 moieties in a time-dependent fashion. The method relies on the controlled infusion of a trifluoroacetic acid/water/acetonitrile solution through the column to cleave the organic functionality (e.g., C8) from the siloxane framework. The bond cleavage solution is reactive enough to cleave the functional groups, even with polar groups embedded within the stationary phase to protect the silica. Both the longitudinal and radial heterogeneity were evaluated by extruding the silica powder into polyethylene tubing and evaluating the percent carbon content in the different sections using thermogravimetric analysis (TGA). TGA analysis shows the presence of a stationary phase gradient in the longitudinal direction but not in the radial direction. Two different gradient profiles were formed with good reproducibility by modifying the infusion method: one exhibited an 'S'-shaped gradient while the other exhibited a steep exponential-like gradient. The gradients were characterized chromatographically using test mixtures, and the results showed varied retention characteristics and an enhanced ability to resolve nicotine analytes.

Impact of four different extraction methods and three different reconstitution solvents on the untargeted metabolomics analysis of human and rat urine samples.

Unsuitable sample preparation may result in loss of important analytes and consequently affect the outcome of untargeted metabolomics. Due to species differences, different sample preparations may be required within the same biological matrix. The study aimed to compare the in-house sample preparation method for urine with methods from literature and to investigate the transferability of sample preparation from human urine to rat urine. A total of 12 different conditions for protein precipitation were tested, combining four different extraction solvents and three different reconstitution solvents using an untargeted liquid-chromatography high resolution mass spectrometry (LC-HRMS) metabolomics analysis. Evaluation was done based on the impact on feature count, their detectability, as well as the reproducibility of selected compounds. Results showed that a combination of methanol as extraction and acetonitrile/water (75/25) as reconstitution solvent provided improved results at least regarding the total feature count. Additionally, it was found that a higher amount of methanol was most suitable for extraction of rat urine among the tested conditions. In comparison, human urine requires significantly less volume of extraction solvent. Overall, it is recommended to systematically optimize both, the extraction method, and the reconstitution solvent for the used biofluid and the individual analytical settings.

Anion-Binding Properties of Short Linear Homopeptides.

A comprehensive thermodynamic and structural study of the complexation affinities of tetra (L1), penta (L2), and hexaphenylalanine (L3) linear peptides towards several inorganic anions in acetonitrile (MeCN) and N,N-dimethylformamide (DMF) was carried out. The influence of the chain length on the complexation thermodynamics and structural changes upon anion binding are particularly addressed here. The complexation processes were characterized by means of spectrofluorimetric, 1H NMR, microcalorimetric, and circular dichroism spectroscopy titrations. The results indicate that all three peptides formed complexes of 1:1 stoichiometry with chloride, bromide, hydrogen sulfate, dihydrogen phosphate (DHP), and nitrate anions in acetonitrile and DMF. In the case of hydrogen sulfate and DHP, anion complexes of higher stoichiometries were observed as well, namely those with 1:2 and 2:1 (peptide:anion) complexes. Anion-induced peptide backbone structural changes were studied by molecular dynamic simulations. The anions interacted with backbone amide protons and one of the N-terminal amine protons through hydrogen bonding. Due to the anion binding, the main chain of the studied peptides changed its conformation from elongated to quasi-cyclic in all 1:1 complexes. The accomplishment of such a conformation is especially important for cyclopeptide synthesis in the head-to-tail macrocyclization step, since it is most suitable for ring closure. In addition, the studied peptides can act as versatile ionophores, facilitating transmembrane anion transport.

Scandium Ion-Promoted Electron-Transfer Disproportionation of 2-Phenyl-4,4,5,5-tetramethylimidazoline-1-oxyl 3-Oxide (PTIO•) in Acetonitrile and Its Regeneration Induced by Water.

2-Phenyl-4,4,5,5-tetramethylimidazoline-1-oxyl 3-oxide (PTIO•), a persistent nitronyl nitroxide radical, has been used for the detection and trapping of nitric oxide, as a redox mediator for batteries, for the activity estimation of antioxidants, and so on. However, there is no report on the reactivity of PTIO• in the presence of redox-inactive metal ions. In this study, it is demonstrated that the addition of scandium triflate, Sc(OTf)3 (OTf = OSO2CF3), to an acetonitrile (MeCN) solution of PTIO• resulted in an electron-transfer disproportionation to generate the corresponding cation (PTIO+) and anion (PTIO-), the latter of which is suggested to be stabilized by Sc3+ to form [(PTIO)Sc]2+. The decay of the absorption band at 361 nm due to PTIO•, monitored using a stopped-flow technique, obeyed second-order kinetics. The second-order rate constant for the disproportionation, thus determined, increased with increasing the Sc(OTf)3 concentration to reach a constant value. A drastic change in the cyclic voltammogram recorded for PTIO• in deaerated MeCN containing 0.10 M Bu4NClO4 was also observed upon addition of Sc(OTf)3, suggesting that the large positive shift of the one-electron reduction potential of PTIO• (equivalent to the one-electron oxidation potential of PTIO-) in the presence of Sc(OTf)3 may result in the disproportionation. When H2O was added to the PTIO•-Sc(OTf)3 system in deaerated MeCN, PTIO• was completely regenerated. It is suggested that the complex formation of Sc3+ with H2O may weaken the interaction between PTIO- and Sc3+, leading to electron-transfer comproportionation to regenerate PTIO•. The reversible disproportionation of PTIO• was also confirmed by electron paramagnetic resonance (EPR) spectroscopy.

Separation of ochratoxins by centrifugal partition chromatography.

The present research work was dedicated to developing an efficient method based on liquid-liquid chromatography (centrifugal partition chromatography, CPC) applicable to routine purifications of ochratoxins (OT) from the liquid culture of the strain A. albertensis SZMC 2107. The crude extract contained numerous components in addition to OTA (90.1 %,) and OTB (1.1 %,) according to HPLC examinations. For the separation of OTs by CPC, several tertiary systems based on acetonitrile, acetone, and short-chain alcohols were examined to find the most applicable biphasic system. The hexane/i-propanol/water 35:15:50 system supplemented with 0.1 % acetic acid was found to be the most efficient for use in CPC separation. Using liquid-liquid instrumental separation, the two OTs, namely OTA (2.23 mg) and OTB (0.031 mg), were successfully isolated with 96.3 % and-72.8 % purity, respectively, from 1 L ferment broth. The identities and purities of the purified components were confirmed and the performance parameters of each separation step and the whole procedure were determined. The developed method could be used effectively to purify OTs for analytical or toxicological applications.

Preparation of high-efficiency HILIC capillary columns utilizing slurry packing at 2100 bar.

Complex mixture analysis requires high-efficiency chromatography columns. Although reversed phase liquid chromatography (RPLC) is the dominant approach for such mixtures, hydrophilic interaction liquid chromatography (HILIC) is an important complement to RPLC by enabling the separation of polar compounds. Chromatography theory predicts that small particles and long columns will yield high efficiency; however, little work has been done to prepare HILIC columns longer than 25 cm packed with sub-2 µm particles. In this work, we tested the slurry packing of 75 cm long HILIC columns with 1.7 µm bridged-ethyl-hybrid amide HILIC particles at 2,100 bar (30,000 PSI). Acetonitrile, methanol, acetone, and water were tested as slurry solvents, with acetonitrile providing the best columns. Slurry concentrations of 50-200 mg/mL were assessed, and while 50-150 mg/mL provided comparable results, the 150 mg/mL columns provided the shortest packing times (9 min). Columns prepared using 150 mg/mL slurries in acetonitrile yielded a reduced minimum plate height (hmin) of 3.3 and an efficiency of 120,000 theoretical plates for acenaphthene, an unretained solute. Para-toluenesulfonic acid produced the lowest hmin of 1.9 and the highest efficiency of 210,000 theoretical plates. These results identify conditions for producing high-efficiency HILIC columns with potential applications to complex mixture analysis.

Detection of trace components in Xiangdan injection of Dalbergia odorifera based on microextraction and back-extraction along with bar-form-diagram strategy.

Xiangdan Injection are commonly used traditional Chinese medicine formulations for the clinical treatment of cardiovascular diseases. However, the trace components of Dalbergia odorifera in Xiangdan Injection pose a challenge for evaluating its quality due to the difficulty of detection. This study proposes a technology combining dispersive liquid-liquid microextraction and back-extraction (DLLME-BE) along with Bar-Form-Diagram (BFD) to address this issue. The proposed combination method involves vortex-mixing tetradecane, which has a lower density than water, with the sample solution to facilitate the transfer of the target components. Subsequently, a new vortex-assisted liquid-liquid extraction step is performed to enrich the components of Dalbergia odorifera in acetonitrile. The sample analysis was performed on HPLC-DAD, and a clear overview of the chemical composition was obtained by integrating spectral and chromatographic information using BFD. The combination of BFD and CRITIC-TOPSIS strategies was used to optimize the process parameters of DLLME-BE. The determined optimal sample pre-treatment process parameters were as follows: 200 µL extraction solvent, 60 s extraction time, 50 µL back-extraction solvent, and 90 s back-extraction time. Based on the above strategy, a total of 29 trace components, including trans-nerolidol, were detected in the Xiangdan Injection. This combination technology provides valuable guidance for the enrichment analysis of trace components in traditional Chinese medicines.

Preparation and evaluation of a piperidinium-sulfonate based zwitterionic monolith for HILIC separation.

In this study, a novel piperidinium-sulfonate based zwitterionic hydrophilic monolith was prepared through thermally initiated co-polymerization of a piperidinium-sulfonate monomer 3-(4-((methacryloyloxy)methyl)-1-methylpiperidin-1-ium-1-yl)propane-1-sulfonate (MAMMPS), and a hydrophilic crosslinker N,N'-methylenebisacrylamide (MBA) using n-propanol and H2O as porogenic system. Satisfactory mechanical and chemical stabilities, good repeatability and high column efficiency (120,000 N/m) were obtained on the optimal monolith. The resulting poly(MAMMPS-co-MBA) monolith showed a typical HILIC retention behavior over an ACN content range between 5 and 95 %. Furthermore, this column exhibited good separation performance for various polar compounds. Compared to quaternary ammonium-sulfonate based zwitterionic hydrophilic monolith, i.e. poly(N,N-dimethyl-N-methacryloxyethyl-N-(3-sulfopropyl)ammonium betaine-co-MBA), the poly(MAMMPS-co-MBA) monolith displayed stronger retention and better selectivity for the tested phenolic and amine compounds at different pH conditions. Finally, this column was applied for the separation of six sulfonamide antibiotics, and the analytical characteristics of the method were evaluated in terms of precision, repeatability, limits of detection (LOD) and quantitation (LOQ). Overall, this study not only developed a novel HILIC monolithic column, but also proved the potential of piperidinium-sulfonate based zwitterionic chemistry as stationary phase, which further increased the structure diversity of zwitterionic HILIC stationary phases.

Computational-aided analysis of the pathway and mechanism of dichloroacetonitrile formation from phenylalanine upon chloramination.

Dichloroacetonitrile (DCAN) is an emerging disinfection by-product (DBP) that is widespread in drinking water. However, the pathway for DCAN formation from aromatic amino acids remains unclear, leading to a lack of an understanding of its explicit fate during chloramination. In this study, we investigated the specific formation mechanism of DCAN during the chloramination of phenylalanine based on reaction kinetics and chemical thermodynamics. The reason for differences between aldehyde and decarboxylation pathways was explained, and kinetic parameters of the pathways were obtained through quantum chemistry calculations. The results showed that the reaction rate constant of the rate-limiting step of the aldehyde pathway with 1.9 × 10-11 s-1 was significantly higher than that of decarboxylation (3.6 × 10-16 s-1 M-1), suggesting that the aldehyde pathway is the main reaction pathway for DCAN formation during the chloramination of phenylalanine to produce DCAN. Subsequently, theoretical calculations were performed to elucidate the effect of pH on the formation mechanism, which aligned well with the experimental results. Dehydrohalogenation was found to be the rate-limiting step under acidic conditions with reaction rate constants higher than those of the rate-limiting step (expulsion of amines) under neutral conditions, increasing the rate of DCAN formation. This study highlights the differences in DCAN formation between the decarboxylation and aldehyde pathways during the chloramination of precursors at both molecular and kinetic levels, contributing to a comprehensive understanding of the reaction mechanisms by which aromatic free amino acids generate DCAN.

Structure-Based Site-Directed Mutagenesis of Hydroxynitrile Lyase from Cyanogenic Millipede, Oxidus gracilis for Hydrocyanation and Henry Reactions.

Hydroxynitrile lyase (HNL) from the cyanogenic millipede Oxidus gracillis (OgraHNL) is a crucial enzyme in the cyanogenesis pathway. Here, the crystal structures of OgraHNL complexed with sulfate, benzaldehyde (BA), (R)-mandelonitrile ((R)-Man), (R)-2-chloromandelonitrile ((R)-2-Cl-Man), and acetone cyanohydrin (ACN) were solved at 1.6, 1.7, 2.3, 2.1, and 2.0 Šresolutions, respectively. The structure of OgraHNL revealed that it belonged to the lipocalin superfamily. Based on this structure, positive variants were designed to further improve the catalytic activity and enantioselectivity of the enzyme for asymmetric hydrocyanation and Henry reactions.

Automated micro-solid-phase extraction clean-up and gas chromatography-tandem mass spectrometry analysis of pesticides in foods extracted with ethyl acetate.

Generic extraction methods for the multi-compound pesticide analysis of food have found their solid place in laboratories. Ethyl acetate and acetonitrile extraction methods have been developed as fast and easy to handle standard multi-compound methods, both feature benefits and limitations. The direct injection to gas chromatography can be impaired by a high burden of coextracted matrix, resulting in deterioration of the chromatographic system and matrix effects, requiring frequent maintenance. Therefore, common clean-up methods, such as dispersive solid-phase extraction, freeze-out of fats, or gel permeation chromatography, have been applied in clean-up. Automated clean-up using micro-solid-phase extraction (µSPE) is a recent development with several demonstrated advantages when employed in the analysis of pesticides and other contaminants in foods extracted with acetonitrile, but it has not yet been evaluated in this application using ethyl acetate for extraction. In this study, an automated procedure using µSPE cartridges was developed and established on an x,y,z robotic sampler for the raw extract clean-up and preparation of diluted samples for injection on a GC-MS/MS system. Validation experiments for 212 pesticides, polychlorinated biphenyls, and polycyclic aromatic hydrocarbons in lettuce, avocado, raspberry, paprika, egg, and liver extracts were performed using µSPE with MgSO4, PSA, C18, and CarbonX. The performance in routine operation is briefly discussed.

Dimethyl carbonate as a green alternative to acetonitrile in reversed-phase liquid chromatography. Part II: Purification of a therapeutic peptide.

Preparative liquid chromatography in reversed phase conditions (RPLC) is the most common approach adopted in the downstream processing for the purification of therapeutic peptides at industrial level. Due to the strict requirements on the quality imposed by the Regulatory Agencies, routinary methods based on the use of aqueous buffers and acetonitrile (ACN) as organic modifier are commonly used, where ACN is practically the only available choice for the purification of peptide derivatives. However, ACN is known to suffers of many shortcomings, such as drastic shortage in the market, high costs and, most importantly, it shows unwanted toxicity for human health and environment, which led it among the less environmentally friendly ones. For this reason, the selection of a suitable alternative becomes crucial for the sustainable downstream processing of peptides and biopharmaceuticals in general. In this paper, a promising green solvent, namely dimethyl carbonate (DMC) has been used for the separation of a peptide not only in linear conditions but also for its purification through non-linear overloaded chromatography. The performance of the process has been compared to that achievable with the common method where ACN is used as organic modifier and to that obtained with two additional solvents (namely ethanol and isopropanol), already used as greener alternatives to ACN. This proof-of-concept study showed that, thanks to its higher elution strength, DMC can be considered a green alternative to ACN, since it allows to reduce method duration while reaching good purities and recoveries. Indeed, at a target purity fixed to 98.5 %, DMC led to the best productivity with respect to all the other solvents tested, confirming its suitability as a sustainable alternative to ACN for the purification of complex biopharmaceutical products.