Non-aqueous Mobile Phase Research Articles

Selective extraction of levoglucosan and its isomers from complex matrices using ligand exchange-solid phase extraction for analysis by liquid chromatography-electrospray ionization-tandem mass spectrometry

The analysis of trace quantities of monosaccharide anhydrides (MAs) in sediments is complicated by the lack of fast and reliable technologies to selectively extract these water-soluble non-ionic compounds from samples of complex composition. Here we describe a solid phase extraction method that takes advantage of the affinity between monosaccharide anhydrides (MAs) and immobilized Na+ ions related to ligand-exchange processes (LE-SPE). The capacity factor of LE-SPE columns was enhanced by using non-aqueous mobile phases such as DCM/MeOH mixtures. We have used the unique properties of LE-SPE columns to selectively extract MAs from lacustrine, coastal, and deep-sea oceanic sediment samples. The analytical procedure produces extracts with low ion suppression effects (0-20%), resulting in ideal conditions for MAs quantification with LC-ESI-MS/MS systems irrespective of the sedimentary matrix and MAs concentration. The analytical method yields repeatable concentration values (RSD of 9-23% for levoglucosan and 15-34% for mannosan and galactosan) and an IS recovery of 45-70%. The instrumental dynamic range is 10-10000 pg injected, but in practice, the methodological lower limit of quantification is constrained by sample contamination during processing. The combination of LE-SPE and LC-ESI-MS/MS has the potential to produce sensitive and reliable technologies to analyze saccharides and amino acids in environmental and biological samples.

Interconversion of nicotine enantiomers during heating and implications for smoke from combustible cigarettes, heated tobacco products, and electronic cigarettes.

Physiological properties of (R)-nicotine have differences compared with (S)-nicotine, and the subject of (S)- and (R)-nicotine ratio in smoking or vaping related items is of considerable interest. A Liquid Chromatography-Mass Spectrometry/Mass Spectrometry (LC-MS/MS) method for the analysis of (S)- and (R)-nicotine has been developed and applied to samples of nicotine from different sources, nicotine pyrolyzates, several types of tobacco, smoke from combustible cigarettes, smoke from heated tobacco products, e-liquids, and particulate matter obtained from e-cigarettes aerosol. The separation was achieved on a Chiracel OJ-3 column, 250 × 4.6 mm with 3-μm particles using a nonaqueous mobile phase. The detection was performed using atmospheric pressure chemical ionization (APCI) in positive mode. The only transition measured for the analysis of nicotine was 163.1 → 84.0. The method has been summarily validated. For the analysis, the samples of tobacco and smoke from combustible cigarettes were subject to a cleanup procedure using solid phase extraction (SPE). It was demonstrated that nicotine upon heating above 450°C for several minutes starts decomposing, and some formation of (R)-enantiomer from a sample of 99% (S)-nicotine is observed. An analogous process takes place when a 99% (R)-nicotine is heated and forms low levels of (S)-nicotine. This interconversion has the effect of slightly increasing the content of (R)-nicotine in smoke compared with the level in tobacco for combustible cigarettes and for heated tobacco products. The (S)/(R) ratio of nicotine enantiomers in e-liquids was identical with the ratio for the particulate phase of aerosols generated by e-cigarette vaping.

Low-cost pencil graphite-based electrochemical detector for HPLC with near-coulometric efficiency

A concentric thin layer cell accommodating a non-standard 0.2 mm diameter pencil graphite disposable working electrode is described. The cell was installed into HPLC manifold as an electrochemical detector. Trouble-free operation in mobile phases containing both low and high content of organic solvents is demonstrated by HPLC analyses of phenolic acids and tocopherol isomers. The data obtained from HPLC separation of model mixtures of gentisic, caffeic and dihydrocaffeic acids show a remarkable electrolytic efficiency exceeding 80% at 200, and 50% at 500 μL min−1, while for gentisic acid the limit of detection (LOD) was 0.4 nmol L−1 at 20 microliter sample loading (8 fmol on-column). Similar performances were found in non-aqueous mobile phase, where a LOD of 0.8 nmol L−1 was achieved for delta-tocopherol. The developed flow-through detector is designed to allow easy replacement of pencil graphite working electrode in a highly reproducible manner. The relative standard deviation for the HPLC analysis of tocopherol isomers was of 5.3% (n = 3, C = 500 nmol L−1). The combination of simple construction, excellent electrochemical performance and hydrodynamics identical to that of commercial UV-VIS HPLC detector suggests that the proposed device is a viable low-cost alternative to commercially available electrochemical detectors.

Isocratic Resolution of Fluoroquinolone-Based Antibiotics on the Phenylethyl-Bonded Phase under Nonaqueous Elution: A Consideration of the Separation Mechanism.

This paper reports the isocratic resolution of 10 fluoroquinolone-based antibiotics and their precursors on the phenylethyl-bonded phase under the elution of the nonaqueous mobile phase composed of acetonitrile, methanol, acetic acid, and triethylamine. Most of the analytes were baseline resolved within 10 minutes. The interaction simulation and Fourier-transform infrared spectroscopy (FTIR) data indicated that the carbonyl-containing group, a secondary or tertiary amine of an analyte, was heavily involved in the retention, resulting in retention with residual silanol groups on the stationary phase. In some cases, the elution reversal or resolution enhancement of analytes was observed when the volume of acidic or basic additive in the mobile phase was dominant. However, the π-π complexation interaction between the fluorine-attached phenyl group of the analyte and the phenylethyl moiety on the stationary phase was not observed. Consequently, the resolution could not be reproduced either on the other stationary phase modified with C18, phenyl, or phenylhexyl moiety under the same chromatographic conditions or under the aqueous elution.

Separation of Isomers of JWH-122 on Porous Graphitic Carbon Stationary Phase with Non-Aqueous Mobile Phase Using Intelligent Software.

Cannabimimetic compounds have gained an increasing attention from the forensic community during the past few years. The present study was aimed to develop a liquid chromatographic separation method for the analysis of JWH-122 and its methyl isomers. In Hungary, JWH-122 is scheduled as a narcotic compound and its methyl isomers fall into the new psychoactive substance category, attracting significantly milder punishment than JWH-122 does. According to our best knowledge, gas chromatography or reversed phase liquid chromatography coupled with mass spectrometry could not be applied for separation and selective determination of methyl-naphthoyl indol isomers. In this study, we aimed to develop a high performance liquid chromatography method with UV and mass spectrometric detection for the separation of JWH-122 and all its possible isomers, depending on the position of methyl group on the naphthyl frame. Different reversed phase columns were used. Alkyl-modified silica with different selectivity and morphology with different mobile phase composition cannot be applied for separation of JWH-122 isomers. Porous graphitic carbon (PGC) column was used for separation of banned JWH-122 and each of its methyl isomers. In method development, a Quality by Designapproach is presented for modeling the retention of the compounds. According to our knowledge, this is the first time reporting the use of intelligent software to estimate the retention on PGC material and using non-aqueous conditions. Retention times predicted by two program packages (STATISTICA® and DryLab®) are compared. The possibilities and limitations of the software modeling in the conditions described above are also evaluated.

Micro Two-Dimensional Thin-Layer Chromatography and Chemometric Analysis of Essential Oils from Selected Mentha Species and Its Application in Herbal Fingerprinting

Micro two-dimensional thin-layer chromatographic systems (micro-2D-TLCs) were optimized using some popular nonaqueous mobile phases on 5 cm × 5 cm silica plates. The optimal 2D TLC systems were chosen based on correlations of RF values vs. mobile phases concentrations performed for 1D separations. Finally plates were developed using the following mobile phases: in the first direction – ethyl acetate/toluene (5/95; v/v) and in the second direction – ethyl acetate/n-heptane (15/85; v/v). Eleven essential oils obtained by hydrodistillation in Deryng apparatus from various mint specimens were separated and then 2D thin-layer chromatograms were densitometrically scanned at the wavelength of 254 nm. Digital images (exported from densitometer software as ASCII files) were processed using ImageJ free software, and then some chemometric parameters were calculated (determination coefficient, congruence coefficient, Euclidean’s distance, Manhattan’s distance, Chebyshev’s distance) and additionally, principal component analysis (PCA) was performed to determine the goodness of matching for individual essential oils. The chemometric analysis allows one to identify individual essential oils and the examined plant materials.

Rapid assessment of the coenzyme Q10 redox state using ultrahigh performance liquid chromatography tandem mass spectrometry.

An improved method for accurate and rapid assessment of the coenzyme Q10 (CoQ10) redox state using ultrahigh performance liquid chromatography tandem mass spectrometry was described, with particular attention given to the instability of the reduced form of CoQ10 during sample preparation, chromatographic separation and mass spectrometric detection. As highly lipophilic compounds in complex biological matrices, both reduced and oxidized forms of CoQ10 were extracted simultaneously from the tissue samples by methanol which is superior to ethanol and isopropanol. After centrifugation, the supernatants were immediately separated on a C18 column with isocratic elution using methanol containing 2 mM ammonium acetate as a non-aqueous mobile phase, and detected by positive electrospray ionization tandem mass spectrometry in multiple reaction monitoring (MRM) mode. Ammonium acetate as an additive in methanol provided enhanced mass spectrometric responses for both forms of CoQ10, primarily due to stable formation of adduct ions [M + NH4](+), which served as precursor ions in positive ionization MRM transitions. The assay showed a linear range of 8.6-8585 ng mL(-1) for CoQ10H2 and 8.6-4292 ng mL(-1) for CoQ10. The limits of detection (LODs) were 7.0 and 1.0 ng mL(-1) and limits of quantification (LOQs) were 15.0 and 5.0 ng mL(-1) for CoQ10H2 and CoQ10, respectively. This rapid extractive and analytical method could avoid artificial auto-oxidation of the reduced form of CoQ10, enabling the native redox state assessment. This reliable method was also successfully applied for the measurement of the CoQ10 redox state in liver tissues of mice exposed to 2,3,7,8-tetrachlorodibenzo-p-dioxin, revealing the down-regulated mitochondrial electron transport chain.

Multiple Response Optimization of a HPLC Method for the Determination of Enantiomeric Purity of S-Ofloxacin

The aim of the study was to develop a new HPLC method for direct chiral separation of Ofloxacin enantiomers using polar non-aqueous mobile phase by application of response surface methodology. Rotatable central composite design (CCD) with eight factorial points, six axial points and six replications in central point was used to evaluate the influence of three independent variables (concentration of methanol, diethylamine and flow rate) on the output responses (capacity factor of first peak, tailing factors of both the enantiomers, resolution between the Ofloxacin enantiomers, retention time of the last peak and chromatographic optimization function). Further, CCD data were combined with multiple response optimization in order to obtain a set of optimal experimental conditions (% methanol/hexane/acetonitrile-43.33/10/46.62 (v/v), % acetic acid/diethylamine-0.4/0.2 and flow rate as 1.4 mL min−1) leading to the most desirable compromise between resolution and analysis time. The method demonstrated good correlation between observed and predicted responses. The developed method was validated according to ICH guidelines and applied for quantitative analysis of two commercially available tablets Zenoflox (Ofloxacin) and Glevo (Levofloxacin). Good agreement was found between the assay results and the label claim of the marketed formulations by showing good %recovery and %CV. The study resulted in a better chromatographic system for the determination of Ofloxacin enantiomers.

Rapid, high performance method for the determination of vitamin K1, menaquinone-4 and vitamin K1 2,3-epoxide in human serum and plasma using liquid chromatography-hybrid quadrupole linear ion trap mass spectrometry

Unlike the other fat-soluble vitamins, vitamin K circulates in the human bloodstream at very low levels because of a low intake in the diet. Mammals have developed an efficient recycling system, known as vitamin K-epoxide cycle, which involve quinone, hydroquinone and epoxide forms of the vitamin. Phylloquinone (K1) is the main homologue, while menaquinone-4 (MK-4) is both a member of the vitamin K2 family and metabolite of K1 in extra-hepatic tissues. Notwithstanding the recent advances, many aspects of the complex vitamin K physiology still remain to be investigated. Therefore, there is a critical need to develop more reliable analytical methods for determining the vitamin K and its metabolites in biological fluids and tissues. Nevertheless, relatively low concentrations, unavailability of some authentic standards and occurrence of interfering lipids make this a challenging task. The method proposed in the present paper can directly and accurately estimate K1, K1 2,3-epoxide (K1O), and MK-4 in human serum and plasma at concentrations in the ng/L–μg/L range, using labelled internal standards and a quadrupole linear ion trap instrument operated in multiple reaction monitoring (MRM) mode. High sensitivity was achieved by removing signal “endogenous suppressors” and making the composition of the non-aqueous mobile phase suitable to support the positive atmospheric pressure chemical ionization of the analytes. An excellent selectivity resulted from the combination of some factors: the MRM acquisition, the adoption of an identification point system, an extraction optimized to remove most of the lipids and a tandem-C18 column-system necessary to separate isobaric interferences from analytes. The method was validated according to the Food and Drug Administration (FDA) guidelines and its accuracy was assessed by analysing 9 samples from the Vitamin K External Quality Assessment Scheme (KEQAS). Its feasibility in evaluating vitamin K status in human serum was also tested by monitoring a group of six healthy subjects and a group of six patients under oral anticoagulant therapy (OAT). Warfarinised patients did not show deficiency of K1 but levels comparable with those of healthy people and an accumulation of K1O up to 3.760μg/L. MK-4 was not detected in either of the two groups.

Monitoring of base catalyzed ethanolysis of Jatropha curcas oil by reversed phase high performance liquid chromatography assisted by ultrasonication

Ultrasonication used for the ethanolysis of non-edible vegetable oil using KOH as a catalyst makes the process fully economic at molar ratio oil to ethanol 1:4, catalyst concentration 0.75wt% of oil, reaction time 7–8min, ultrasonic amplitude 50% (100W/m3) and cycle 0.7s. ultrasonication reduces the reaction time comparing to the conventional batch process. RP-HPLC was used to monitor the reactivity during the base catalyzed ethanolysis of Jatropha curcas oil. A RP-HPLC method has been developed for the determination of triacylglycerol, diacylglycerol, monoacylglycerol as well as free fatty acids and their corresponding ethyl esters. The determination of these components was carried out using a 40min combined linear gradient with aqueous-organic and non-aqueous mobile phase steps: 90% methanol+10% water in 10min, 100% methanol in 0min, 60% methanol+15% n-hexane+25% propan-2-ol in 30min and 90% methanol+10% water for last 10min was used for fast monitoring of transesterification reaction. Individual calibration curves (relative peak area versus amount of the compound) were found for quantitative analysis of ethyl esters and acylglycerol shows good linearity at UV detection 215nm. The identification of each compound can be done by APCI-MS in the position ion mode.

Comparison of nonaqueous hydrophilic interaction chromatography with aqueous normal‐phase chromatography on hydrosilated silica‐based stationary phases

We investigated the retention behavior of phenolic acids in nonaqueous normal-phase (NP) LC with buffered methanol/acetonitrile mobile phases on hydrosilated silica-based stationary phases. The silica hydride, Diamond hydride, Bidentate C18, and Cholesterol columns showed a higher retention of phenolic acids in the nonaqueous mobile phases than in aqueous NP mobile phases. There are some selectivity differences between the aqueous and nonaqueous mobile phases, but generally the resolution and selectivity are better in the aqueous systems. The retention of the phenolic acids tested decreased with increasing concentration of methanol in the mobile phase, up to 20% v/v methanol. At increased temperatures, the retention factors and peak widths decrease in both NP modes, showing linear ln k versus 1/T plots, due to a single retention mechanism over the temperature range from 25°C up to the column stability limit, however, the best separations are achieved at low temperatures. The enthalpic and entropic contributions to the retention were determined, and the differences between the aqueous and nonaqueous modes are possibly due to the adsorbed water layer.

Two-dimensional thin-layer chromatographic determination of phenolic antioxidants fromEupatorium cannabinumextracts on cyano-bonded polar stationary phases

Two-dimensional thin layer chromatography on cyano-bonded polar stationary phase was performed to optimize the separation of some antioxidant phenolic compounds from Eupatorium cannabinum extracts. Propan-2-ol mixed with n-heptane and ethyl acetate mixed with n-heptane were used as non-aqueous mobile phases in normal phase separations (1st direction of development in 2-dimensional high-performance thin-layer chromatography (2D-HPTLC mode)) and methanol mixed with water was used as a mobile phase in reversed phase (2nd direction of development in 2D-HPTLC mode). The plates were sprayed by use of Merck TLC sprayer using 2-(diphenylboryoxy)-ethylamine and PEG4000 (Merck, Darmstadt, Germany) or DPPH and photographed in CAMAG Cabinet UV lamp at 254 nm and 365 nm by use of Fuji 8 mpx camera. The plots RF non-aqueous mobile phase vs RF aqueous mobile phase were prepared to find optimal orthogonal 2D-TLC systems for the separation of investigated test compounds and then extracts from Eupatorium cannabinum. Satisf...

Two-dimensional Thin Layer Chromatographic Separation of Phenolic Compounds from Eupatorium cannabinum Extracts and their Antioxidant Activity

Two dimensional thin layer chromatography on DIOL polar bonded stationary phase was performed to optimize the separation of some anioxidant phenolic compounds from Eupatorium cannabinum extracts. Ethyl-methyl ketone mixed with n-heptane and ethyl acetate mixed with n-heptane were used as non-aqueous mobile phases in normal phase separations (1st direction of development in 2D-HPTLC mode) and methanol mixed with water was used as a mobile phase in reversed phase (2nd direction of development in 2D-HPTLC mode). The plates were sprayed by use of Merck TLC sprayer using 2-(diphenylboryoxy)-ethylamine and PEG4000 (Merck, Darmstadt, Germany) or DPPH and photographed in Camag Cabinet UV lamp at 254 nm and 365 nm by use of Fuji 8 mpx camera. Satisfactory separations of antioxidant phenolic compounds in Eupatorium cannabinum extracts were obtained by use of optimized 2D-HPTLC systems.

Fast liquid chromatography-diode array detection assisted by chemometrics for quantification of seven ultraviolet filters in effluent wastewater

A fast chromatographic method is presented for simultaneous quantification of seven organic ultraviolet (UV) filters (benzophenone-3,4-methylbenzilidene camphor, octocrylene, 1-(4-tert-butylphenyl)-3-(4-methyoxyphenyl)1,3-propanedione), ethylhexyl methoxy cinnamate, ethylhexyl salicylate and homosalate) in effluent wastewater samples. The UV filters were pre-concentrated by Bond Elut-ENV cartridges and separated on an ODS column (15cm×0.46cm, 5μm) in less than 2.5min using a non-aqueous mobile phase of methanol–acetonitrile (50:50, v/v) with flow-rate of 1.5mLmin−1. Appropriate baseline correction through asymmetric least squares was applied to reduce the matrix of background signals in three way data. Then, second-order calibration based on multivariate curve resolution-alternating least squares (MCR-ALS) was implemented on the unfolded three-way data obtained from liquid chromatography with diode array detection (LC-DAD) through standard addition calibration method for handling co-eluted peaks, systematic and proportional errors. Recoveries ranging from 76% to 130% and %RSD values less than 11.2 for all UV filter shows the accuracy and precision of the proposed method in wastewater samples. In addition, statistical t-test as well as computed elliptical joint confidence region (EJCR) confirms the accuracy of the proposed method and indicates the absence of both constant and proportional errors in the predicted concentrations. This study demonstrates that coupling of the fast HPLC-DAD method with powerful algorithm of MCR-ALS can be considered as an efficient method for quantification of UV filters in highly contaminated samples of wastewaters where both time and cost per each analysis can be reduced significantly.

2D TLC separation of phenols by use of RP-18 silica plates with aqueous and non-aqueous mobile phases

Two-dimensional (2D) TLC of eleven phenols has been performed on octadecyl-silica. The most efficient systems were selected by analysis of retention data obtained by one-dimensional chromatography using aqueous and non-aqueous mobile phases. Correlation graphs were plotted to illustrate the separation in the chromatographic systems examined. Complete separation of the phenols was achieved by using an aqueous mobile phase in the first dimension and a non-aqueous mobile phase in the second dimension. Statistical methods (principal-components analysis and cluster analysis) were used for better characterization of the TLC systems.

LC–MS Analysis of Sertraline and Its Active Metabolite in Human Serum Using a Silica Column with a Non-Aqueous Polar Mobile Phase

A sensitive liquid chromatography–mass spectrometry method for the simultaneous determination of sertraline (SER) and its major metabolite norsertraline (NOR) from serum was developed and validated in the context of a pharmacokinetic study in pregnant women. The separations were achieved on a silica column with a non-aqueous polar mobile phase consisting of acetonitrile, methanol and ammonium acetate at a flow rate of 0.5 mL min−1. The concentrations were measured using a single quadruple mass spectroscopic detector supplied with atmospheric pressure ionization electrospray. Sample preparation consisted of a simple liquid–liquid procedure. The detector was set in selective ion mode for each compound of interest, 306 m/z for SER and 275 m/z for NOR. Calibration curves were generated by least square linear regression for concentration of 5–160 ng mL−1 for SER and from 10 to 320 ng mL−1 for NOR. The curves for both compounds of interest were linear, with correlation coefficients r2 ≥ 0.999.

Effect of the vapour phase on the TLC separation of tropane alkaloids

Summary The effect of conditioning the silica layer by mobile phase vapour or ammonia vapour on the retention of alkaloids eluted with non-aqueous mobile phases was examined. The effect of vapour phase composition on system efficiency, peak symmetry, and separation selectivity were also investigated. The most effective and selective systems were used for separation of the alkaloid fraction of Datura innoxia.

Development and validation of a LC method for the enantiomeric purity determination of S-ropivacaine in a pharmaceutical formulation using a recently commercialized cellulose-based chiral stationary phase and polar non-aqueous mobile phase

Ropivacaine is the first enantiomerically pure long-acting local anaesthetic used for surgical anaesthesia and post-operative pain relief.A liquid chromatographic (LC) method using acetonitrile as the main solvent and cellulose tris(4-chloro-3-methylphenylcarbamate) coated on silica as chiral stationary phase was successfully developed and applied for the enantiomeric purity determination of S-ropivacaine in a pharmaceutical formulation (Naropin®). The key role played by the acidic additive (trifluoroacetic acid or formic acid) in the enantioseparation of basic drugs in these LC systems was demonstrated by the reversal of ropivacaine enantiomers elution order observed when both acids were compared. In order to elute the enantiomeric impurity (R-ropivacaine) before S-ropivacaine, formic acid (FA) was selected. The temperature and the percentages of acidic additive and hexane in the mobile phase were found to significantly influence the retention and resolution of these enantiomers. The optimized mobile phase consisted of ACN/0.1% DEA/0.2% FA/5% hexane (v/v/v/v). The temperature was set at 35°C to avoid the interference from a peak system related to the presence of water in the sample on ropivacaine enantiomers.The LC method was then fully validated applying the strategy based on total measurement error and accuracy profiles. The accuracy profile obtained by linear regression after square root transformation was selected, the acceptance limits being settled at ±10% for the intended use of this analytical method. The relative bias was lower than 1.5%, while the RSD values for repeatability and intermediate precision were both below 1.0%. The limit of detection (LOD) and the limit of quantification (LOQ) were found to be about 0.2 and 1.0μg/mL, respectively, corresponding to 0.02 and 0.1% of the enantiomeric impurity in S-ropivacaine.

Effect of the vapour phase on the separation of isoquinoline alkaloids by thin-layer chromatography

Summary The effect of conditioning of the silica layer by mobile phase vapour, diethylamine vapour and its aqueous and methanolic solutions, and ammonia vapour on the retention of alkaloids eluted with multicomponent non-aqueous mobile phases was examined. Effect of conditioning time and vapour phase composition on system efficiency and peak symmetry was investigated, as also was the effect of vapour phase composition on separation selectivity. The most effective and selective systems were used for separation of the alkaloid fraction from Fumaria officinalis.

Optimization of the LC enantioseparation of chiral pharmaceuticals using cellulose tris(4-chloro-3-methylphenylcarbamate) as chiral selector and polar non-aqueous mobile phases

The resolving power of a new commercial polysaccharide-based chiral stationary phase, Sepapak-4, with cellulose tris(4-chloro-3-methylphenylcarbamate) coated on silica microparticles as chiral selector, was evaluated toward the enantioseparation of ten basic drugs with widely different structures and hydrophobic properties, using ACN as the main component of the mobile phase. A multivariate approach (experimental design) was used to screen the factors (temperature, n-hexane content, acidic and basic additives) likely to influence enantioresolution. Then, the optimization was performed using a face-centered central composite design. Complete enantioseparation could be obtained for almost all tested chiral compounds, demonstrating the high chiral discrimination ability of this chiral stationary phase using polar organic mobile phases made up of ACN and containing an acidic additive (TFA or formic acid), 0.1% diethylamine and n-hexane. These results clearly illustrate the key role of the nature of the acidic additive in the mobile phase.