High-performance Liquid Chromatographic Separation Research Articles

Identification of 2-Hydroxymethyl-3,6-diethyl-5-methylpyrazine as a Key Retronasal Burnt Flavor Compound in Soy Sauce Aroma Type Baijiu Using Sensory-Guided Isolation Assisted by Multivariate Data Analysis.

The burnt flavor (overcooked rice flavor) is often retronasally perceived during the soy sauce aroma type Chinese Baijiu (Chinese distilled spirit) tasting and regarded as an important quality marker of the Baijiu. The sensory-guided isolation approach assisted by multivariate data analysis was applied to identify the compounds responsible for the retronasal burnt flavor in the Baijiu. Using two-dimensional offline high performance liquid chromatography separation and taste dilution analysis, a subfraction with the highest burnt flavor intensity was isolated from the Baijiu sample. In the subfraction, six potential flavor compounds were detected by ultra performance liquid chromatography-mass spectrometry. The most probable burnt flavor compound was screened by projection to latent structure regression analysis of the correlation between the potential compounds and their impact on burnt flavor. The target compound was identified as 2-hydroxymethyl-3,6-diethyl-5-methylpyrazine by means of high-resolution mass spectrometry and chemical synthesis. Sensory evaluation revealed a relatively low burnt flavor threshold of 2.89 μmol/L in 53% ethanol aqueous solution. On the basis of the dose-overthreshold factor and addition experiment, this compound was confirmed as the key contributor to the burnt flavor of soy sauce aroma type Baijiu.

QUANTITATIVE ASSAY OF ASPIRIN AND (SALICYLIC ACID AND HEAVY METALS AS IMPURATIES) IN IRAQI’S MARKET ASPIRIN TABLETS USING DIFFERENT ANALYTICAL METHODS

Objective: Easy and precise methods were developed for estimation of aspirin (ASP), impurities from such as salicylic acid (SAL) and heavy metal ions (HMI) in ASP tablets that available in the Iraqi’s market using High-performance liquid chromatography (HPLC), UV–VIS spectrophotometry and atomic absorption spectrophotometric (AAS).Methods: HPLC separation was carried out using C18 as stationary phase and acetonitrile (ACN): water in the ratio of (10: 90 v/v) as a mobile phase for HPLC method and as a solvent for UV-VIS spectrophotometric for quantitative ASP and SAL at 254 nm for HPLC, 226 and 296 nm for UV measurements. AAS was used for HMI determination.Results: ASP and SAL gave absorbance maxima at 226 and 296 nm in ACN: H2O solvent. The Beer’s law was obeyed in the range of 0.05-20 for ASP and 0.02-8 µg/ml for SAL. Correlation coefficients (R2) were 0.9996 and 0.9992 for ASP and SAL respectively, for HPLC and LOD value was 0.006 for ASP and 0.004 μg/ml for SAL. The % recovery for the developed method was found to be in the range of (98.80 to 101.26%) and (98.67 to 103.33%) for ASP and SAL respectively, within the acceptable range, that approved by world health organization (WHO).Conclusion: The proposed method can help research studies, quality control and routine analysis with lesser resources available. The results of the assay of pharmaceutical formulation of the developed method are highly reliable and reproducible and is in good agreement with the label claim of the medicines.

Quantitative assessment of the metabolic products of iron oxide nanoparticles to be used as iron supplements in cell cultures

Iron nanoparticles (NPs) metabolism is directly associated to human health due to their use as anemia treatment and should be studied in detail in cells. Here we present a speciation strategy for the determination of the metabolic products of iron oxide nanoparticles coated by tartaric and adipic acids in enterocytes-like cell models (Caco-2 and HT-29). Such methodology is based on the use of SDS-modified reversed phase high performance liquid chromatography (HPLC) separation using inductively coupled plasma-mass spectrometry (ICP-MS) as Fe selective detector. Post-column isotope dilution analysis is used as quantification tool by adding 57Fe as isotopically enriched standard. To assess the separation capability of the method, two different iron nanostructures: iron sucrose nanoparticles -Venofer®- used as model suspension and iron tartrate/adipate-modified nanoparticles, both of about 4 nm (core size) were evaluated. The two nanostructures were injected into the system showing good peak profiles and quantitative elution recoveries (>80%) in both cases. In addition, both nanoparticulate fractions could be based-line separated from ionic iron species, which needed to be complexed with 1 mM citrate to elute from the column. Exposed cells up to 0.5 mM of iron tartrate/adipate-modified nanoparticles were specifically treated to extract the internalized NPs and the extracts examined using the proposed strategy. The obtained results revealed the presence of three different fractions corresponding to nanoparticle aggregates, dispersed nanoparticles and soluble iron respectively in a single chromatographic run. Quantitative experiments (column recoveries ranging from 60 to 80%) revealed the presence of the majority of the Fe in the nanoparticulated form (>75%) by summing up the dispersed and aggregate particles. Such experiments point out the high uptake and low solubilization rate of the tartrate/adipate NPs making these structures highly suitable as Fe supplements in oral anemia treatments.

Graphene oxide as a stationary phase for speciation of inorganic and organic species of mercury, arsenic and selenium using HPLC with ICP-MS detection.

This work demonstrates the power of graphene and graphene oxide (GO) as stationary phases for high performance liquid chromatography (HPLC) based elemental speciation analysis combined with detection by inductively coupled plasma mass spectrometry (ICP-MS). Inorganic mercuric and organomercuric compounds coordinate with 2-thiosalicylic acid (TSA) to form Hg-TSA complexes. These complexes are retained by GO owing to its strong π electron stacking capability for TSA. Separation of the four mercury species tested was achieved within 12min with resolutions of 1.8-3.4. Similarly, inorganic anionic species of arsenic and selenium, and organoarsenicals are electrostatically attracted by aromatic quaternary ammonium cations in the mobile phases. Organoarsenicals also can be separated by using long alkyl quaternary ammonium compounds. Aromatic quaternary ammonium compounds possess particularly high affinity to GO because of strong π interaction. This leads to effective retention of the As/Se anions. A comparison between graphene and GO as stationary phases for HPLC separation of mercury and arsenic species demonstrates negligible difference. Arsenic species are separated within 32min, and selenium species are achieved within 20min. The mobile phase also allows efficient separation of iodate, iodide, bromate, bromide, chromic acid and chromate. Analysis of a certified fish tissue by HPLC-ICP-MS using the GO@SiO2 column demonstrates its feasibility for routine elemental analysis. Good agreement is found between experimental results and certified values, with recoveries ranging between 92 and 96%. Graphical abstract Graphene oxide as a stationary phase for high performance liquid chromatography with inductively coupled plasma mass spectrometric detection (HPLC-ICP-MS) in speciation analyses of mercury, arsenic, selenium, iodine, bromine and chromium is achieved for the first time.

Analysis of alkaloids from Peganum harmala L. sequential extracts by liquid chromatography coupled to ion mobility spectrometry

An orthogonal two dimensional analysis method based on high performance liquid chromatography (HPLC) separation and electrospray ionization-ion mobility spectrometry (ESI-IMS) detection was developed for the analysis of alkaloid compounds from Peganum harmala L. seeds. Reverse phase (RP) and hydrophilic interaction chromatography (HILIC) were compared for the most optimal performance using three different chromatographic columns. The experimental results suggest that HILIC mode is a better option for combining with the ESI-IMS system for higher sensitivity and ease in hyphenating. Under optimized conditions, alkaloids from different extraction phases were determined by means of the established HPLC-IMS method. More compounds from Peganum harmala L. seed extracts were differentiated on the HPLC-ESI-IMS system by their retention time and drift time than by HPLC or ESI-IMS alone, and thirteen alkaloids were tentatively identified based on m/z and fragment ions using ultra-high-performance liquid chromatography tandem mass-spectrometry (UPLC-MS/MS). Hence, our results indicate that this method can be considered to be advantageous over traditional absorbance detection methods for resolving complex mixtures because of complementary separation steps, elevated peak capacity, and higher sensitivity.

Simultaneous determination of total homocysteine, cysteine, glutathione, and N-acetylcysteine in brain homogenates by HPLC.

We have developed a simple, fast, accurate, and cheap method for the simultaneous determination of total cysteine, homocysteine, glutathione, and N-acetylcysteine in brain homogenates based on the reduction of disulfide bonds by tris(2-carboxyethyl) phosphine, pre-column derivatization of free thiol groups with 2-chloro-1-methylquinolinium tetrafluoroborate followed by ion-pair reversed-phase high-performance liquid chromatography separation with ultraviolet detection. The separation of thiol derivatives was achieved in 10min. Linearity was observed in the range of 10-300, 0.7-10, 2-30, and 3-20μmol/L homogenate with a limit of detection of 3.7, 0.2, 0.8, and 1.2μmol/L homogenate for cysteine, homocysteine, glutathione, and N-acetylcysteine, respectively. The precision, calculated as relative standard deviation, was in the range of 1.21-4.77, 1.53-14.35, 0.47-1.92, and 1.61-8.95% for cysteine, homocysteine, glutathione, and N-acetylcysteine, respectively. The presented method was successfully applied to the selective determination of total amino thiols in pig brain tissue samples.

Preparation and Chromatographic Features of Fibrous Core–Shell HPLC Packing Material

In this study, fibrous core–shell silica particles were successfully synthesized via a one-step oil–water biphase stratification coating strategy. The core–shell silica particles were composed of 3-µm non-pore silica cores and thin shells (50–100 nm), which have radial-like direct channels and a large pore size (19.89 nm). The fibrous core–shell silica particles were further modified by n-octadecyltrichlorosilane and used as stationary-phase media in high-performance liquid chromatography (HPLC). The chromatographic properties of the particles were systematically studied in small-molecule and protein separation processes. The results showed that the back pressure was as low as 8.5 MPa under the 1.0-mL min−1 flow velocity. Furthermore, fibrous core–shell silica particles with an 80-nm shell were used for separating seven small molecules within 10 min and six proteins within 6 min. This work demonstrates that the fibrous core–shell silica particles could be used as an HPLC stationary phase with good performance and low back pressure, and that they have great potential for application to HPLC separation in the future.

Analysis and Identification of Polyphenolic Compounds in Green Foods Using a Combination of HPLC-ESI-IT-TOF-MS/MS

Abstract The beneficial effects of green foods on human health are well known. These benefits are mainly due to polyphenol content. The aim of this work was the development and utilization of an advanced mass spectrometry (MS) technique combined with high-performance liquid chromatography (HPLC) for the analysis and identification of polyphenolic compounds in green foods. HPLC-ESI-MS/MS analyses of selected extracts prepared from various types of green food (Urtica dioica L. and Allium ursinum L.) and their polyphenolic profiles were performed by an LCMS-IT-TOF analyzer equipped with electrospray ionization. HPLC separation was conducted by a Kinetex XB-C18 column (100µ2.1 mm; 2.6 µm) using gradient elution (water + 0.1% formic acid: acetonitrile + 0.1% formic acid) with a flow rate of 0.2 mL/min. The MS1-MS3 analyses were performed within a 50-1000 m/z range in both positive and negative ionization modes. The total analysis time was 20 minutes and injected volumes were 2 µL and 5 µL, respectively

Determination of major phenolic compounds in apples: Part I-Optimization of high-performance liquid chromatography separation with diode array detection.

The separation of seven phenolic compounds including gallic acid, chlorogenic acid, epicatechin, quercitrin, rutin, phloridzin, and phloretin present in apple peel and pulp and differing in elution properties has been optimized using high-performance liquid chromatography with diode array detection. Several stationary phases were tested to achieve the efficient separation of phenolic compounds in fruit extracts and C18 was found to be the most efficient. Core-shell and fully porous C18 packings were assessed with respect to the complex composition of the fruit extracts. The developed high-performance liquid chromatography method comprised gradient elution in which mobile phase A was water at pH 2.8 adjusted with acetic acid and B was acetonitrile. The gradient shape was the following: 0min 95% A/5% B, 2.5min 85% A/15% B, 12min 50% A/50% B, 15min 95% A/5% B. The flow rate was 1mL/min, injection volume 10μL, and UV detection at 255, 280, 320, and 365nm was applied. Our method was validated for both C18 core-shell and fully porous packings. The resolution 6.2-14.8, symmetry 0.99-1.34, peak capacity 18-60, peak area repeatability 0.45-1.00% relative standard deviation, calibration range 0.125-5mg/mL (0.25-10mg/mL for chlorogenic acid and rutin), correlation coefficients of calibration curve 0.9976-0.9997, and accuracy evaluated as recovery 95.56-107.54% were determined for the core-shell column.

Hybridization of metal–organic framework and monodisperse spherical silica for chromatographic separation of xylene isomers

Abstract Metal–organic frameworks (MOFs) packed in the column have been a promising candidate as the stationary phase for high performance liquid chromatography (HPLC). However, the direct packing of irregular MOF powder could raise some problems like high back pressure and low column efficiency in the HPLC separation. In this work, UiO-66 capable of separating xylenes was supported effectively on the surface of the monodisperse spherical silica microspheres by one-pot method. The hybridization of UiO-66 and silica microspheres (termed UiO-66@SiO2 shell–core composite) was prepared by stirring the suspension of the precursors of UiO-66 and COOH terminated silica in the N,N-dimethylformamide with heating. The shell–core composite material UiO66@SiO2 was characterized by SEM, TEM, PXRD and FTIR. Then, it was used as a packing material for the chromatographic separation of xylene isomers. Xylene isomers including o-xylene, m-xylene and p-xylene were efficiently separated on the column with high resolution and good reproducibility. Moreover, the UiO-66@SiO2 shell–core composites packed column still remained reverse shape selectivity as UiO-66 possessed, and the retention of xylenes was probably ascribed to the hydrophobic effect between analytes and the aromatic rings of the UiO-66 shell. The UiO-66@SiO2 shell–core composites obtained in this study have some potential for the separation of structural isomers in HPLC.

Pseudomonas sp. ST4 produces variety of active compounds to interfere fungal sexual mating and hyphal growth.

SummarySexual mating of compatible sporida is essential for Sporisorium scitamineum to form dikaryotic mycelia and then cause infection on sugarcane. Our previous work identified a Pseudomonas sp. ST4 from a soil sample, which showed a promising biocontrol potential by inhibiting the mating of S. scitamineum sporida and hyphal growth. In this study, we set to isolate the active compounds from Pseudomonas sp. ST4 through solid fermentation. High‐performance liquid chromatography (HPLC) separation coupling with bioassay showed that Pseudomonas sp. ST4 produced a range of antimicrobial compounds. Two of the major components were purified following acetate extraction, silica gel and HPLC separation. Nuclear magnetic resonance (NMR) and liquid chromatography–mass spectrometry (LC‐MS) analysis identified these active compounds are 4‐hydroxybenzaldehyde and indole‐3‐carbaldehyde respectively. Further analysis showed that the former compound only inhibited the hyphal growth of the fungus at a concentration of 3 mM, while the latter interfered the fungal sexual mating at a concentration of 0.6 mM and affected hyphal growth at a concentration of 2 mM. Treatment of corn plants with 3 mM indole‐3‐carbaldehyde significantly inhibited corn smut infection, with a control rate up to 94%. Further analysis of the structure and activity relationship revealed that indole has a much stronger inhibitory activity against the fungal sexual mating than indole‐3‐carbaldehyde. The results from this study provide new agents for control and prevention of the sugarcane smut disease, and the active compounds could also be used to probe the molecular mechanisms of fungal sexual mating.

Reliable radiosynthesis of 4-[10B]borono-2-[18F]fluoro-l-phenylalanine with quality assurance for boron neutron capture therapy-oriented diagnosis

ObjectiveThe aim of this study was to establish a reliable and routine method for the preparation of 4-[10B]borono-2-[18F]fluoro-l-phenylalanine (l-[18F]FBPA) for boron neutron capture therapy-oriented diagnosis using positron emission tomography.MethodsTo produce l-[18F]FBPA by electrophilic fluorination of 4-[10B]borono-l-phenylalanine (l-BPA) with [18F]acetylhypofluorite ([18F]AcOF) via [18F]F2 derived from the 20Ne(d,α)18F nuclear reaction, several preparation parameters and characteristics of l-[18F]FBPA were investigated, including: pre-irradiation for [18F]F2 production, the carrier F2 content in the Ne target, l-BPA-to-F2 ratios, separation with high-performance liquid chromatography (HPLC) using 10 different eluents, enantiomeric purity, and residual trifluoroacetic acid used as the reaction solvent by gas chromatography-mass spectrometry.ResultsThe activity yields and molar activities of l-[18F]FBPA (n = 38) were 1200 ± 160 MBq and 46–113 GBq/mmol, respectively, after deuteron-irradiation for 2 h. Two 5 min pre-irradiations prior to [18F]F2 production for 18F-labeling were preferable. For l-[18F]FBPA synthesis, 0.15–0.2% of carrier F2 in Ne and l-BPA-to-F2 ratios > 2 were preferable. HPLC separations with five of the 10 eluents provided injectable l-[18F]FBPA without any further formulation processing, which resulted in a synthesis time of 32 min. Among the five eluents, 1 mM phosphate-buffered saline was the eluent of choice. The l-[18F]FBPA injection was sterile and pyrogen-free, and contained very small amounts of D-enantiomer (< 0.1% of l-[18F]FBPA), l-BPA (< 1% of l-FBPA), and trifluoroacetic acid (< 0.5 ppm).Conclusionsl-[18F]FBPA injection was reliably prepared by the electrophilic fluorination of l-BPA with [18F]AcOF followed by HPLC separation with 1 mM phosphate-buffered saline.

Synchronised determination of chlorogenic acid and five flavonoids in mulberry leaves using HPLC with photodiode array detection

An analytical method based on high-performance liquid chromatography separation with diode array detection was developed and validated for the synchronised determination of chlorogenic acid and five flavonoids (rutin, isoquercitrin, quercitrin, quercetin, and luteolin) in mulberry leaves (Morus spp.). Chromatographic separation was carried out under gradient elution conditions on a Shim Pack VP-ODS C18 (250 × 4.6 mm, particle size 5 μm) column at a temperature of 30 °C using a mobile phase consisting of 0.5% (v/v) phosphoric acid in water and acetonitrile at a flow rate of 0.9 ml/min. The column eluent process was best at 330 nm (for chlorogenic acid, quercitrin, and luteolin), 350 nm (for rutin and isoquercitrin), and 365 nm (for quercetin). Application of optimum extraction conditions led to extraction of chlorogenic acid and five flavonoids from mulberry leaves with mean recoveries of 97.78-103.24%. The developed method was validated in terms of linearity, recovery, precision, and stability. The relati...

Structural Characterisation of the Stress Degradation Products of Adefovir Dipivoxil by LC-MS and NMR

Hepatitis B is one of the most common infectious disease in the world. Adefovir dipivoxil (ADL) is the recently approved drug that is prominently used in the treatment of hepatitis B. The present work focuses on the development of a stability indicating assay method for the separation and charecterization of the stress degradation products of ADL. The drug degraded under the hydrolytic (acid, alkaline and neutral) and oxidative stress conditions resulting in the formation of two degradation products. The High Performance Liquid Chromatography (HPLC) separation was achieved using C18 stationary phase (150 x 4.6 mm x 5μm) and mobile phase composed of 10 mM ammonium acetate as a buffer while acetonitrile as an organic modifier in gradient elution mode. The degradation products were characterised by tandem mass spectrometry consisting of quadrupole and time-of-flight mass analysers. The major degradation product (DP 2) of hydrolytic stress was isolated and its chemical structure was confirmed using various NMR techniques (1H, 13C, COSY, DEPT 135 and HSQC). The proposed method can be used for quantitative and qualitative determination of ADL during the process and formulation development stages.

An inline ion-exchange system in a chemiluminescence-based analyzer for direct analysis of N-nitrosamines in treated wastewater

A newly developed, ion exchange-based inline pretreatment system was used to mitigate the effect of background constituents in natural water and treated wastewater to achieve rapid, reliable, and sensitive analysis of N-nitrosamines. The pretreatment system (anion exchange module, AEM) was incorporated into a high-performance liquid chromatograph (HPLC) coupled with a photochemical reactor (PR) and chemiluminescence (CL) detector (HPLC-PR-CL), which can analyze four hydrophilic N-nitrosamines at ng/L levels. This system requires no pre-concentration of the water sample nor the use of deuterated surrogates, unlike other conventional N-nitrosamine analytical techniques. The AEM converted anions in the eluent to hydroxide ions after HPLC separation and increased eluent pH, allowing for the subsequent photochemical reactions, which are otherwise achieved by pH conditioning with an additional dosing pump of basic chemical. The AEM also removed anionic interfering compounds (e.g. nitrate) from the samples, allowing for improved N-nitrosamine analysis in treated wastewater. The operating conditions of the AEM and PR were optimized to obtain sensitive and stable analytical performance. As a result, the lowest-concentration minimum reporting levels of N-nitrosodimethylamine, N-nitrosomorpholine, N-nitrosomethylethylamine, and N- nitrosopyrrolidine using the optimized system were 0.42, 0.54, 0.58, and 1.4 ng/L, respectively. The improved analytical method was validated by comparing the results with a conventional method based on gas chromatography coupled with a mass spectrometric ion trap detector. These results indicated that HPLC-PR-CL equipped with an inline AEM can be competitively applied as a rapid analytical technique for the determination of N-nitrosamines in various water matrices.

High Performance Liquid Chromatography Separation of Epigenetic Cytosine Variants.

Epigenetic modifications enable cells to genetically respond to chemical inputs from environmental sources. These marks play a pivotal role in normal biological processes (e.g., differentiation, host defense and metabolic programs) but also contribute to the development of a wide variety of pathological conditions (e.g., cancer and Alzheimer’s disease). In particular, DNA methylation represents very stable epigenetic modification of cytosine bases that is strongly associated with a reduction in gene activity. Although High Performance Liquid Chromatography (HPLC) methodologies have been used to resolve methylated cytosine from unmodified cytosine bases, these represent only two of the five major cytosine analogs in the cell. Moreover, failure to resolve these other cytosine analogs might affect an accurate description of the cytosine methylation status in cells. In this present study, we determined the HPLC conditions required to separate the five cytosine analogs of the methylation/demethylation pathway. This methodology not only provides a means to analyze cytosine methylation as a whole, but it could also be used to more accurately calculate the methylation ratio from biological samples.

Arsenic speciation in hair and nails of acute promyelocytic leukemia (APL) patients undergoing arsenic trioxide treatment

Arsenic in hair and nails has been used to assess chronic exposure of humans to environmental arsenic. However, it remains to be seen whether it is appropriate to evaluate acute exposure to sub-lethal doses of arsenic typically used in therapeutics. In this study, hair, fingernail and toenail samples were collected from nine acute promyelocytic leukemia (APL) patients who were administered intravenously the daily dose of 10 mg arsenic trioxide (7.5 mg arsenic) for up to 54 days. These hair and nail samples were analyzed for arsenic species using high performance liquid chromatography separation and inductively coupled plasma mass spectrometry detection (HPLC-ICPMS). Inorganic arsenite was the predominant form among water-extractable arsenicals. Dimethylarsinic acid (DMAV), monomethylarsonic acid (MMAV), monomethylarsonous acid (MMAIII), monomethylmonothioarsonic acid (MMMTAV), and dimethylmonothioarsinic acid (DMMTAV) were also detected in both hair and nail samples. This is the first report of the detection of MMAIII and MMMTAV as metabolites of arsenic in hair and nails of APL patients.

Separation of enantiomers of new psychoactive substances by high-performance liquid chromatography.

New psychoactive substances are defined as compounds with consciousness-changing effects and have been developed simultaneously with classical drugs. They arise through structural modifications of illegal substances and are mainly produced to circumvent laws. Availability is simple, since new psychoactive substances can be purchased from the Internet. Among them many chemical drug compound classes are chiral and thus the two resulting enantiomers can differ in their effects. The aim of this study is to develop a suitable chiral high-performance liquid chromatography separation method for a broad spectrum of new psychoactive substances using cellulose tris(3,5-dichlorophenylcarbamate) as a chiral selector. Experiments were performed by high-performance liquid chromatography in normal-phase mode under isocratic conditions using ultraviolet detection. Direct separation was carried out on a high-performance liquid chromatography column (Lux® i-Cellulose-5, 3.5 μm, Phenomenex®), available since 2016. Excellent separation results were obtained for cathinones. After further optimization, even 47 instead of 39 out of 52 cathinones showed baseline separation. For amphetamine derivatives, satisfactory results were not achieved. Further, new psychoactive substances from other compound classes such as benzofuranes, thiophenes, phenidines, phenidates, morpholines, and ketamines were partially resolved, depending on the polarity and degree of substitution. All analytes, which were mainly purchased from the Internet, were proven to be traded as racemates.

Cellulose tris-(3,5-dimethylphenylcarbamate)-based chiral stationary phase for the enantioseparation of drugs in supercritical fluid chromatography: comparison with HPLC.

A cellulose tris-(3,5-dimethylphenylcarbamate)-based chiral stationary phase was studied as a tool for the enantioselective separation of 21 selected analytes with different pharmaceutical and physicochemical properties. The enantioseparations were performed using supercritical fluid chromatography. The effect of the mobile phase composition was studied. Four different additives (diethylamine, triethylamine, isopropylamine, and trifluoroacetic acid) and isopropylamine combined with trifluoroacetic acid were tested and their influence on enantioseparation was compared. The influence of two different mobile phase co-solvents (methanol and propan-2-ol) combined with all the additives was also evaluated. The best mobile phase compositions for the separation of the majority of enantiomers were CO2 /methanol/isopropylamine 80:20:0.1 v/v/v or CO2 /propan-2-ol/isopropylamine/trifluoroacetic acid 80:20:0.05:0.05 v/v/v/v. The best results were obtained from the group of basic β-blockers. A high-performance liquid chromatography separation system composed of the same stationary phase and mobile phase of similar properties prepared as a mixture of hexane/propan-2-ol/additive 80:20:0.1 v/v/v was considered for comparison. Supercritical fluid chromatography was found to yield better results, i.e. better enantioresolution for shorter analysis times than high-performance liquid chromatography. However, examples of enantiomers better resolved under the optimized conditions in high-performance liquid chromatography were also found.

Stability-Indicating HPLC and HPTLC Methods for Determination of Agomelatine and its Degradation Products.

Two accurate, sensitive and highly selective stability-indicating methods are developed and validated for simultaneous determination of Agomelatine (AGM) and its forced degradation products (Deg I and II). The first method is High-Performance Liquid Chromatography for separation and quantitation of AGM, Deg I and II on a C18 column (250 mm × 4.6 mm, 5 μm p.s) in isocratic mode by using a binary mixture of Potassium dihydrogen phosphate (0.05 M, pH adjusted to 2.9 with orthophosphoric acid): acetonitrile (60:40, v/v) at a flow rate of 2 mL/min. The components were detected at 230 nm over a concentration range of 0.5-10 μg/mL for AGM and 0.5-5 μg/mL for both Deg I and II. The second method is High-Performance Thin-Layer Chromatography, where AGM, Deg I and II were separated on silica gel HPTLC F254 plates using chloroform:methanol:ammonia solution (9:1:0.1, by volume) as a developing system. The separated bands were scanned at 230 nm over the concentration range of 0.2-1.2 μg/band for AGM in pure form and human plasma and 0.1-1 μg/band for both Deg I and II. The proposed methods were successfully applied for analysis of AGM in pharmaceutical formulations. The results obtained by the proposed methods were statistically compared to the reported HPLC method revealing high accuracy and good precision.