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.

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.

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.

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.

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.

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.

Electronic relaxation mechanism of 9-methyl-2,6-diaminopurine and 2,6-diaminopurine-2'-deoxyribose in solution.

Prolonged ultraviolet exposure results in the formation of cyclobutane pyrimidine dimers (CPDs) in RNA. Consequently, prebiotic photolesion repair mechanisms should have played an important role in the maintenance of the structural integrity of primitive nucleic acids. 2,6-Diaminopurine is a prebiotic nucleobase that repairs CPDs with high efficiency when incorporated into polymers. We investigate the electronic deactivation pathways of 2,6-diaminopurine-2'-deoxyribose and 9-methyl-2,6-diaminopurine in acetonitrile and aqueous solution to shed light on the photophysical and excited state properties of the 2,6-diaminopurine chromophore. Evidence is presented that both are photostable compounds exhibiting similar deactivation mechanisms upon the population of the S1 (ππ* La ) state at 290 nm. The mechanism involves deactivation through the C2- and C6-reaction coordinates and >99% of the excited state population decays through nonradiative pathways involving two conical intersections with the ground state. The radiative and nonradiative lifetimes are longer in aqueous solution compared to acetonitrile. While τ1 is similar in both derivatives, τ2 is ca. 1.5-fold longer in 2,6-diaminopurine-2'-deoxyribose due to a more efficient trapping in the S1 (ππ* La ) minimum. Therefore, 2,6-diaminopurine could have accumulated in significant quantities during prebiotic times to be incorporated into non-canonical RNA and play a significant role in its photoprotection.

Improved sample introduction approach in hydrophilic interaction liquid chromatography to avoid breakthrough of proteins.

When therapeutic proteins are analysed under hydrophilic interaction liquid chromatography (HILIC) conditions, there is an inherent mismatch between the sample diluent (proteins must be solubilised in aqueous media) and the mobile phase, which is mostly composed of aprotic solvent (acetonitrile). This difference in eluent strength between sample diluent and mobile phase is responsible for severe analyte breakthrough and peak distortion. As demonstrated with therapeutic proteins of different sizes (insulin of 6 kDa, anakinra of 17 kDa and rituximab subunits of 25 and 50 kDa), only very small volumes of 0.1-0.2 µL can be injected without breakthrough effects, when performing rapid analysis on short HILIC columns of 20-50 mm, leading to poor sensitivity. In order to avoid the undesired effect of the strong sample diluent, a special injection program should be preferred. This consists in the addition and automatic injection of a defined volume of weak solvent (acetonitrile) along with the sample to increase retention factors during sample loading. Various injection programs were tested, including the addition of a pre-injection or post-injection or both (bracketed injection) of acetonitrile plugs. Several weak to strong injection solvent ratios of 1:1, 1:2, 1:4 and 1:10 were tested. Our work proves that the addition of a pre-plug solvent with a weak vs. strong injection solvent ratio of 1:10 is a valuable strategy to inject relatively large volumes of proteins in HILIC, regardless of column dimensions, thus maximising sensitivity. No peak deformation or breakthrough was observed under these conditions. However, it is important to note that peak broadening (40 % larger peaks) was observed when the injection program increased the injection solvent ratio from 1:1 to 1:10. Finally, this strategy was applied to a wide range of therapeutic mAb products with different physico-chemical properties. In all cases, relatively large volumes can be successfully injected onto small volume HILIC columns using a purely aqueous sample diluent, as long as an appropriate (weak) solvent pre-injection is applied.

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.

Implementation of Quality by Design Approaches for Development and Validation of Reverse-Phase High-Performance Liquid Chromatography Assay Method for Determination of Glycyrrhizin in Nanoformulation.

Glycyrrhizin (GL) is the principal constituent of Glycyrrhiza glabra, having antiallergic, anticancer, anti-inflammatory, and antimicrobial action. The reverse-phase high-performance liquid chromatography (RP-HPLC) analytical method was used to quantitatively estimate GL in a nanoformulation and validated as per International Conference on Harmonization Q2 (R1) standards. A stationary phase of the C18-HL reversed-phase column and a mobile phase of acetonitrile and water were used for effective elution. The chromatographic conditions of RP-HPLC were optimized utilizing a quality-by-design approach to accomplish the required chromatographic separation of GL from its nanoformulation with minimal experimental runs. Optimized RP-HPLC conditions for the assay method consist of acetonitrile (41%) and water, pH 1.8, balanced with phosphoric acid (0.1%) as a mobile phase with a flow rate of 1 mL/min. The retention time was found at 7.25 min, and method validation confirmed its sensitivity, preciseness, accuracy, and robustness.

Dimethyl carbonate as a green alternative to acetonitrile in reversed-phase liquid chromatography. Part I: Separation of small molecules.

Nowadays, environmental problems are drawing the attention of governments and international organisations, which are therefore encouraging the transition to green industrial processes and approaches. In this context, chemists can help indicate a suitable direction. Beside the efforts focused on greening synthetic approaches, currently also analytical techniques and separations are under observation, especially those employing large volumes of organic solvents, such as reversed-phase liquid chromatography (RPLC). Acetonitrile has always been considered the best performing organic modifier for RPLC applications, due to its chemical features (complete miscibility in water, UV transparency, low viscosity etc); nevertheless, it suffers of severe shortcomings, and most importantly, it does not fully comply with Environmental, Health and Safety (EHS) requirements. For these reasons, alternative greener solvents are being investigated, especially easily available alcohols. In this work, chromatographic performance of the most common solvents used in reversed-phase chromatography, i.e., acetonitrile, ethanol and isopropanol, have been compared to a scarcely used solvent, dimethyl carbonate (DMC). The analytes of interest were two small molecules, caffeine and paracetamol, whose kinetics and retention behaviour obtained with the four solvents have been compared, and all contributions to band broadening have been assessed. Results about kinetic performance are very promising, indicating that a small amount (7 % v/v) of DMC is able to produce the same efficiency as a 2.5-times larger ACN volume (18 % v/v), and larger efficiency than alcohols. This paper reports, for the first time, fundamental studies concerning the mass transfer phenomena when DMC is used as an organic solvent in RPLC, and, together with the companion paper, represents the results of a research whose final aim was to discover whether DMC is suitable for chromatographic applications both in linear and preparative conditions.

Dynamic headspace GC-MS/MS analysis of ethylene oxide and 2-chloroethanol in dry food commodities: a novel approach.

With frequent RASFF notifications from the EU countries, the residue testing of ethylene oxide (EtO) and its metabolite 2-chloroethanol (2-CE) in food commodities has become essential to check their compliance with MRLs. This study, for the first time, aimed at establishing a dynamic headspace-GC-MS/MS method for the simultaneous determination of these two analytes in acetonitrile extracts of cumin, ashwagandha, chilli powder, turmeric powder, guar gum, locust bean gum, and ginger powder. The samples (4 g) were extracted using acetonitrile (10 mL). A dispersive-solid phase extraction cleanup step with primary secondary amine sorbent (50 mg/mL) reduced the interfering signal of (matrix-derived) acetaldehyde by >40% in chilli powder, ginger, turmeric, and guar gum. This cleanup was not required for sesame seeds. With high selectivity and sensitivity, the GC-MS/MS approach identified and quantified both compounds simultaneously. At the spiking levels of 0.01, 0.02, and 0.05 mg/kg, the recoveries and precision were satisfactory (70-120%, RSDs, ≤15%). The headspace method-performance was similar to liquid injections. The method provided reproducible results when evaluated by two different laboratories. The method provided high-precision results for incurred residue analysis. Given its efficiency, the validated method is anticipated to improve the effectiveness of monitoring of EtO residues in food commodities.

Direct extraction with acetonitrile of hemp seed oil for the analysis of pesticides by using comprehensive two-dimensional gas chromatography-triple quadrupole mass spectrometry.

The method herein described involves a rapid and limited-volume (0.5 mL of acetonitrile) solvent-extraction sample preparation process, for pesticide determination in hemp seed oil. The extraction method was characterized by the absence of both clean-up or pre-concentration steps. The extracts were directly analyzed through cryogenic-modulation comprehensive two-dimensional gas chromatography coupled to triple quadrupole mass spectrometry. The novelty characterizing the present research [compared to a previous one (Arena et al., 2023)] is related to the extension of the number of pesticides (97), and to the investigation of a more challenging matrix, contained in a vegetable oil of increasing interest among consumers. Linearity, limits of detection and quantification, accuracy, precision, recovery, and matrix effect were measured. Particular emphasis was devoted to the matrix effect, with the co-extracted matrix amount defined. Three international regulations (Canada, California, Europe) were considered, and the obtained limits of quantification were found to be too high in five (Canada) and twelve (Europe) cases, for a total number of 15 pesticides. The analysis of ten commercial samples showed the presence of seven pesticide residues in four of them, at concentration levels ranging from 0.02 to 0.98 mg kg-1, with most over the regulation residue limits.

Development and validation of a sensitive ultra-high-performance liquid chromatography-tandem mass spectrometry method for quantitative analysis of bambermycin in livestock and aquatic products: Implications for food safety control and regulatory enforcement.

A sensitive and accurate analytical method was developed and validated to detect bambermycin, a commonly used antibiotic in animal feed and livestock. The presence of bambermycin residues in food products can pose health risks to consumers, emphasizing the need for a sensitive and accurate analytical method. A reversed-phase analytical column was utilized with a mobile phase comprising 0.005 mol/L ammonium acetate in 5% acetonitrile (A) and 0.005 mol/L ammonium acetate in 95% acetonitrile (B) to achieve effective chromatographic separation. Quantitative determination of bambermycin in various samples, including beef, pork, chicken, milk, eggs, flatfish, eel, and shrimp, was performed using ultra-high-performance liquid chromatography-tandem mass spectrometry. Sample extraction involved a mixture of methanol and a 25% ammonium hydroxide solution, followed by low-temperature purification and phospholipid removal utilizing a Phree cartridge. The method exhibited a satisfactory recovery rate ranging from 69% to 100%. Validation results demonstrated the reliability, robustness, and accuracy of the method, exhibiting good linearity, precision, and recovery. This validated method can be applied for routine analysis of bambermycin residues, assisting in the development of effective monitoring and control measures to ensure the safety of livestock and aquatic products.

Towards an accurate method for column void volume determination using liquid chromatography-mass spectrometry.

Prediction of analyte retention times requires prior knowledge of the column void volume, the measurement of which is still highly contested within the literature and therefore experimental based prediction is often used. In this study, we investigated deuterated acetonitrile as an isotopically labelled mobile phase component to observe its elution behaviour in a binary mixture with water at 25 different mobile phase compositions (from 5 to 95 vol.% of acetonitrile), on two stationary phases (C8 and C18), and at two temperatures (30 and 40 °C) using LC-MS. The same experimental design was additionally used for three commonly used neutral void volume markers: uracil, phloroglucinol and N,N-dimethylformamide. Temperature was observed to influence the elution of acetonitrile in an inversely proportional manner with higher temperatures coinciding with lower elution times. By utilizing a three-way ANOVA, the composition of the mobile phase has been shown to have a significant effect on deuterated acetonitrile and other investigated void volume markers, demonstrating the fact that both void volume markers and acetonitrile itself exhibit retention-like behaviour. Excess adsorption isotherms for acetonitrile were calculated using deuterated acetonitrile elution data. The comparison of void volumes, obtained with conventional neutral void volume markers, revealed the former to be 24-36% lower than the void volume obtained using deuterated acetonitrile, as an isotopically labelled mobile phase component. For a water:acetonitrile mobile phase, the minor disturbance method using deuterated acetonitrile to obtain an integral average void volume (2.08 and 2.05 mL for C18 at 30 and 40 °C, respectively and 2.16 and 2.13 mL for C8 at 30 and 40 °C, respectively) was found to be the most appropriate method for determining the elusive column void volume.

Resolving phenylephrine HCl and guaifenesin enantiomers on cellulose-based chiral stationary phases: Separation of four enantiomers on 50-mm column.

Chiral high performance liquid chromatographic technique usually employs polysaccharide-based stationary phases in a normal phase mode. This frequently generates large waste of organic solvents. Using shorter columns of 50 mm length as well as a mobile phase with a high water percentage are common approaches for greening this analytical technique. In this context, a new chiral chromatographic technique was developed for simultaneous enantio-separation of phenylephrine HCl and guaifenesin racemates. Four 50 mm cellulose-based columns were experimented to separate the four enantiomers in a reversed phase mode. A face centered design was then employed to optimize the mobile phase acetonitrile% and flow rate on Lux Cellulose-1 (50 × 4.6 mm, 5 µm). The simultaneous resolution of the cited drugs enantiomers was achieved using acetonitrile-water (30:70, by volume), with a flow rate of 0.5 ml min-1 . These optimized chromatographic conditions separate the enantiomers in 7 min running time, generating about 1.0 ml acetonitrile per run. The proposed method was favorably compared with other reported chiral ones in terms of waste volume generated and analysis time required.

Capacitively coupled contactless conductivity detection to account for system-induced gradient deformation in liquid chromatography.

The time required for method development in gradient-elution liquid chromatography (LC) may be reduced by using an empirical modelling approach to describe and predict analyte retention and peak width. However, prediction accuracy is impaired by system-induced gradient deformation, which can be especially prominent for steep gradients. As the deformation is unique to each LC instrument, it needs to be corrected for if retention modelling for optimization and method transfer is to become generally applicable. Such a correction requires knowledge of the actual gradient profile. The latter has been measured using capacitively coupled "contactless" conductivity detection (C4D), featuring a low detection volume (approximately 0.05 µL) and compatibility with very high pressures (80 MPa or more). Several different solvent gradients, from water to acetonitrile, water to methanol, and acetonitrile to tetrahydrofuran, could be measured directly without the addition of a tracer component to the mobile phase, exemplifying the universal nature of the approach. Gradient profiles were found to be unique for each solvent combination, flowrate, and gradient duration. The profiles could be described by convoluting the programmed gradient with a weighted sum of two distribution functions. Knowledge of the exact profiles was used to improve the inter-system transferability of retention models for toluene, anthracene, phenol, emodin, sudan-I and several polystyrene standards.

Managing sample introduction problems in hydrophilic interaction liquid chromatography.

Sample injection can cause serious problems in hydrophilic interaction liquid chromatography (HILIC) when the injection solvent has higher elution strength than the mobile phase (mp). It can lead to asymmetric peak shapes and poor efficiency. The problem can occur when the mp contains a high proportion of organic e.g. 95% acetonitrile (a weak solvent) whereas the injection solvent contains a higher proportion of water (a strong solvent) that is necessary to dissolve polar samples. We investigated different strategies to overcome this problem. A simple method is pre-column dilution where the injector is programmed to deliver a plug of weak solvent (e.g. pure acetonitrile) along with the sample dissolved in a solvent with higher water content than the mp. Another option is to use alternative organic solvents to acetonitrile in the injection solvent, e.g. isopropanol, acetone or tetrahydrofuran, that may give enhanced sample solubility. The role of the volume of injection solvents was investigated as well as the possible effects of mass overload on the results. The use of small sample volumes is always recommended to reduce mismatch effects.