Coupling Of Thin-layer Chromatography Research Articles

Coupling thin layer chromatography with mass spectrometry for detection and identification of sertraline and its metabolite in the urine

The aim of the study was to detect sertraline and its metabolites in urine under standardized thin layer chromatography (TLC) screening conditions and identify the metabolites using the mass spectrometry method. Materials and methods. Urine samples, collected within 30 hours in portions of 20–50 mL, commenced from the seventh hour after the administration of a single therapeutic dose of the drug. The sample preparation process involved dilute acid hydrolysis, followed by the extraction of the native compound and metabolites with chloroform at a pH of 8–9. Thin layer chromatography studies of the extracts were conducted using four unified TLC systems recommended by The International Association of Forensic Toxicologists for general drug screening. Chromatograms were subjected to color reactions with a variety of chromogenic reagents. For the analysis of eluates from chromatograms, a Varian 1200 L mass spectrometer (Netherlands) equipped with a dual quadrupole mass analyzer was employed. Identification was performed through direct sample introduction into the ion chamber, electron-impact ionization (70 eV), and full ion scanning mode. Results. The spot of the native drug on the chromatogram was identified by the Rf, value. Metabolite of sertraline was identified as N-desmethyltrihydroxysertraline by the molecular ion peak in the mass spectrum. Conclusions. The study demonstrated the ability of TLC to detect sertraline and its metabolite, N-desmethyltrihydroxysertraline, in urine after the administration of a single therapeutic dose of the drug. The chromatographic mobility of the native compound and N-desmethyltrihydroxysertraline in the unified TLC screening systems, along with the results of their visualization using chromogenic reagents for toxicological drug screening, was determined. Furthermore, the potential of coupling TLC with mass spectrometry for the separation, detection, and confirmatory identification of sertraline and its metabolic products in urine was established.

TLC-Freeze SERS: APowerful Tool for Monitoring Synthetic Reactions.

Thin layer chromatography (TLC) is widely used to confirm the formation of the target compound in chemical synthesis. The key issue in TLC is spot identification relying on retention factors. Coupling of TLC with surface-enhanced Raman spectroscopy (SERS), which gives direct molecular information, is a possible choice, to overcome this challenge. However, interference from the stationary phase and impurities on the nanoparticles added for SERS measurements significantly degrades TLC-SERS efficiency. We have found that freezing effectively eliminates such interferences and dramatically improves the performance of TLC-SERS. This study shows that TLC-freeze SERS is a powerful tool for monitoring essential organic reactions. This method can identify the product and side-products of similar structures, detect compounds with high sensitivity, and provide information of quantities that allows the reliable determination of the reaction time based on kinetic analysis.

Separation and determination of the group-type composition of modern base and lubricating oils with a wide range of polarity, especially emitted to the environment

Lubricating oils are composed of base oils (>85% v/v) and enriching additives (<15% v/v). Three types of base oils may be distinguished: 1) traditional bases (obtained by low-volatile fractions from crude oil distillation refining), 2) synthetic bases (mainly poly-alpha-olefins, sometimes esters, especially succinic acid esters), 3) bases of natural origin (especially obtained from refined plant oils). The bases of natural origin are the only ones recommended for application when lubricating oil may be emitted to the environment (e.g. when the machine with an open cutting system is used). Group-type separation and analysis of group-type composition of base and lubricating oils are of significant importance in quality control and environmental monitoring. Due to the potentially wide range of polarity of the components of base and lubricating oils, group- type separation becomes a difficult separation problem. It is also a serious analytical problem due to the considerable diversity of physicochemical properties. The authors propose a new procedure for the separation and determination of the group-type composition of base and lubricating oils using thin-layer liquid chromatography in normal phase systems (abr. NP-TLC) on silica gel plates impregnated with berberine salt/in the coupling of thin-layer chromatography with flame ionization detection (abr. TLC-FID). A new, effective procedure of TLC plates impregnation with berberine sulphate was presented. The proposed procedure ensures the visualization of all groups of base oils. Extensive experimental research showed that a 2-step development procedure with application of n-hexane up to 100% height of development +15 min and further n-hexane: isopropanol: tri-fluoroacetic acid 96.25: 3: 0.75 (v: v: v) up to 75% height of development is advantageous for the group-type separation, both in TLC-FID and TLC.

Mass spectrometry imaging of thin-layer chromatography plates using laser desorption/low-temperature plasma ionisation.

Thin-layer chromatography (TLC) is a classic method for the separation and analysis of complex mixtures. Biological assays, chemical derivatisation and spectroscopy techniques are compatible with TLC and provide extra information about isolated compounds. However, coupling TLC to mass spectrometry is hampered by the difficulty to desorb the analytes from the silica surfaces. In this study, we used a multimodal ion source for laser desorption (LD) and low-temperature plasma (LTP) post-ionisation. Efficient desorption was reached by covering the TLC plates with activated carbon. Regions of interest can be analysed by spots, by lines or by area. We show the separation of methylxanthines from coffee, tea and cocoa preparations by TLC, with subsequent mass spectrometry imaging (MSI). Using a lateral resolution of 400 μm × 400 μm, allowed the acquisition of 21 895 spectra in 2.4 h (2.5 pixels per s). Further, we demonstrate the possibility of direct mass fragmentation studies and quantification. We mounted the system on an Open LabBot with a theoretical lateral resolution of 12.5 μm and performed the visualisation of ions of interest and the pixel-wise review of mass spectra with our free software RmsiGUI (). This non-proprietary and modular platform enables the cost-efficient adaption of the system and further development by the community.

Characterization of additives in NR formulations by TLC-IR (UATR)

It is a well-established fact that rubber accelerator is essential to provide solution in different sectors. However, there is a reversal process which can reduce the material performance. Sulfur accelerators donors and organic peroxides have been presented as a solution to the problem. The methodology development that can separate or characterize those components is a challenge and still allows gaps, explained by the application of conventional technique to reach this goal. This study aimed at contributing to the use of off-line coupling of thin layer chromatography (TLC)/infrared spectroscopy (IR) by Universal Attenuated Total Reflection (UATR) for analysis of N-cyclohexyl-2-benzotiazolsulfenamide (CBS), tetraethylthiuram disulfide (TMTD) and dicumyl peroxide (DCP), in natural poly-cis-isoprene (NR) formulations, containing naphthenic oil. The best results were obtained for the plasticizer and DCP, in formulations that had a greater proportion of these compounds. The separation of CBS and TMTD was made with less effectiveness, due to bands overlapping.

Laser desorption-ion mobility spectrometry as a useful tool for imaging of thin layer chromatography surface

We present a novel method for coupling thin layer chromatography (TLC) with ion mobility spectrometry (IMS) using laser desorption technique (LD). After separation of the compounds by TLC, the TLC surface was sampled by the LD-IMS without any further manipulation or preparation. The position of the laser was fixed and the TLC plate was moved in desired directions by the motorized micro-positioning stage. The method was successfully applied to analyze the TLC plates containing explosives (tri nitro toluene, 1,3,5-trinitro- 1,3,5-triazacyclohexane, pentaerythritol tetranitrate, 2,4-dinitro toluene and 3,4-dinitro toluene), amino acids (alanine, proline and isoleucine), nicotine and diphenylamine mixtures and detection limits for these compounds were determined. Combination of TLC with LD-IMS technique offers additional separation dimension, allowing separation of overlapping TLC analytes. The time for TLC sampling by LD-IMS was less than 80s. The scan rate for LD is adjustable so that fast and effective analysis of the mixtures is possible with the proposed method.

Simultaneous Determination of Size and Quantification of Gold Nanoparticles by Direct Coupling Thin layer Chromatography with Catalyzed Luminol Chemiluminescence.

The increasing use of metal-based nanoparticle products has raised concerns in particular for the aquatic environment and thus the quantification of such nanomaterials released from products should be determined to assess their environmental risks. In this study, a simple, rapid and sensitive method for the determination of size and mass concentration of gold nanoparticles (AuNPs) in aqueous suspension was established by direct coupling of thin layer chromatography (TLC) with catalyzed luminol-H2O2 chemiluminescence (CL) detection. For this purpose, a moving stage was constructed to scan the chemiluminescence signal from TLC separated AuNPs. The proposed TLC-CL method allows the quantification of differently sized AuNPs (13 nm, 41 nm and 100 nm) contained in a mixture. Various experimental parameters affecting the characterization of AuNPs, such as the concentration of H2O2, the concentration and pH of the luminol solution, and the size of the spectrometer aperture were investigated. Under optimal conditions, the detection limits for AuNP size fractions of 13 nm, 41 nm and 100 nm were 38.4 μg L−1, 35.9 μg L−1 and 39.6 μg L−1, with repeatabilities (RSD, n = 7) of 7.3%, 6.9% and 8.1% respectively for 10 mg L−1 samples. The proposed method was successfully applied to the characterization of AuNP size and concentration in aqueous test samples.

Quantitative Characterization of Gold Nanoparticles by Coupling Thin Layer Chromatography with Laser Ablation Inductively Coupled Plasma Mass Spectrometry.

Metal nanoparticles (NPs) determination has recently attracted considerable attention because of the continuing boom of nanotechnology. In this study, a novel method for separation and quantitative characterization of NPs in aqueous suspension was established by coupling thin layer chromatography (TLC) with laser ablation inductively coupled plasma mass spectrometry (LA-ICP-MS). Gold nanoparticles (AuNPs) of various sizes were used as the model system. It was demonstrated that TLC not only allowed separation of gold nanoparticles from ionic gold species by using acetyl acetone/butyl alcohol/triethylamine (6:3:1, v/v) as the mobile phase, but it also achieved the separation of differently sized gold nanoparticles (13, 34, and 47 nm) by using phosphate buffer (0.2 M, pH = 6.8), Triton X-114 (0.4%, w/v), and EDTA (10 mM) as the mobile phase. Various experimental parameters that affecting TLC separation of AuNPs, such as the pH of the phosphate buffer, the coating of AuNPs, the concentrations of EDTA and Triton X-114, were investigated and optimized. It was found that separations of AuNPs by TLC displayed size dependent retention behavior with good reproducibility, and the retardation factors (R(f) value) increased linearly with decreasing nanoparticle size. The analytical performance of the present method was evaluated under optimized conditions. The limits of detection were in the tens of pg range, and repeatability (RSD, n = 7) was 6.3%, 5.9%, and 8.3% for 30 ng of 13 nm AuNPs, 34 nm AuNPs, and 47 nm AuNPs, respectively. The developed TLC-LA-ICP-MS method has also been applied to the analysis of spiked AuNPs in lake water, river water, and tap water samples.

Surface-enhanced Raman spectroscopy of the anti-cancer drug irinotecan in presence of human serum albumin

The development of nanotechnological devices and their clinical application in medicine has become increasingly important, especially in the context of targeted and personalized therapy. This is particularly important in cancer therapy, where antitumor drugs are highly cytotoxic and often exert their therapeutic effect at concentrations close to systemic toxicity. In the last years a growing number of studies has started to report the use of plasmonic nanoprobes in the field of theranostics, broadening the application of vibrational spectroscopies like Raman scattering and surface enhanced Raman scattering (SERS) in biomedicine. The present work aims to identify and characterize the vibrational profiles of a widely used anticancer drug, irinotecan (CPT-11). With a rational approach, SERS experiments have been performed on this analyte employing both Au and Ag colloids, starting from simple aqueous solutions up to albumin mixtures. A major step forward for drug detection in albumin solutions has been taken with the adoption of a simple deproteinization strategy, and a two-in-one-step separation and identification by coupling thin layer chromatography, TLC, with SERS (TLC-SERS). The latter has revealed to be a valid system for protein separation and simultaneous analyte detection, showing a potential to become an innovative, sensitive and low cost method for antineoplastic drug profiling in patients’ body fluids.

Thin-layer chromatography combined with diode laser thermal vaporization inductively coupled plasma mass spectrometry

Here we present a novel coupling of thin-layer chromatography (TLC) to diode laser thermal vaporization inductively coupled plasma mass spectrometry (DLTV ICP MS). DLTV is a new technique of aerosol generation which uses a diode laser to induce pyrolysis of a substrate. In this case the cellulose stationary phase on aluminum-backed TLC sheets overprinted with black ink to absorb laser light. The experimental arrangement relies on economic instrumentation: an 808-nm 1.2-W continuous-wave infrared diode laser attached to a syringe pump serving as the movable stage. Using a glass tubular cell, the entire length of a TLC separation channel is scanned. The 8-cm long lanes were scanned in ∼35s. The TLC – DLTV ICP MS coupling is demonstrated on the separation of four cobalamins (hydroxo-; adenosyl-; cyano-; and methylcobalamin) with limits of detection ∼2pg and repeatability ∼15% for each individual species.

Direct TLC/MALDI–MS coupling for modified polyamidoamine dendrimers analyses

Polyamidoamine (PAMAM) are synthetic dendrimers which present attractive properties for the biological and biomedical fields, as they proved to be efficient drug and gene carriers. In order to increase their transfection efficiency, chemical modifications of the amino end-groups had been reported. In this work, the synthesis of the ammonia-cored G1(N) PAMAM and the consecutive chemical modification with glycine or phenylalanine amino-acids were monitored using the coupling of thin layer chromatography (TLC) with matrix–assisted laser desorption ionization–mass spectrometry (MALDI–MS). Thus, the monitoring of the PAMAM synthesis included the identification of the by-products such as defective structures of PAMAM dendrimers as well as the study of phenylalanine-grafted PAMAM oligomer distribution.

Comprehensive thin-layer chromatography mass spectrometry of flavanols from Juniperus communis L. and Punica granatum L.

The coupling of thin-layer chromatography with mass spectrometry (TLC–MS) for the analysis of monomeric flavanols and proanthocyanidins in samples presented as complex matrices has been studied. The elution conditions for TLC–MS were optimised and full scans were compared with selected reaction monitoring for the MS detection of compounds. The performance of silica gel and cellulose plates with different developing solvents in TLC–MS was assessed. Cellulose plates provided superior sensitivity while ionisation suppression was encountered with silica plates. The use of a HILIC guard column beyond the elution head was found to facilitate detection of monomer compounds on silica plates. A new comprehensive TLC×MS procedure for screening flavanols in the entire chromatogram was developed as an alternative to the use of 4-dimethylaminocinnamaldehyde to determine the locations of compounds on the plate. This new procedure was applied to detect flavanols in the peel of Punica granatum L. fruits and in seeds of Juniperus communis L., in which flavanols and proanthocyanidin dimers and trimers were detected for the first time.

TLC‐SERS study of Syrian rue (Peganum harmala) and its main alkaloid constituents

Surface‐enhanced Raman spectroscopy (SERS) has found increasing acceptance in art conservation and forensic science for its great potential in detecting trace amounts of material. However, SERS is not a separation technique, and, therefore, it is not always suitable for distinguishing different components in a mixture. Coupling of thin layer chromatography (TLC) and SERS has been investigated in this article as a tool for the separation and identification of four alkaloids, namely harmalol, harmaline, harmane and harmine, from the seed extract of Syrian rue (Peganum harmala). The alkaloids contained in this plant were historically used as a dye and for medicinal purposes and have recently drawn attention due to their antitumor activity. The use of TLC over high‐performance liquid chromatography (HPLC) is a convenient way to reduce the amount of material, equipment and time needed for the analysis, and coupling of TLC with SERS provides vibrational information on each compound in the mixture. HPLC analyses with diode array detection were also carried out as a test of our technique, to ascertain the composition of Syrian rue extract and validate the results obtained from TLC‐SERS investigations. In addition, Syrian rue extract and its commercial alkaloid components were characterized by SERS and normal Raman spectroscopy here for the first time, in order to provide valuable reference data to be used for identification purposes. Copyright © 2012 John Wiley &amp; Sons, Ltd.

Application of TLC and LA ICP SF MS for speciation of S, Ni and V in petroleum samples

A coupling of thin layer chromatography with laser ablation ICP SF MS was developed for the fractionation of Ni, V, Fe and S in crude oil and its fractions (saturate, aromatic, resin and asphaltene). The detection limits were 18ngg−1 and 23ngg−1 for nickel and vanadium, respectively, and a sample could be characterized in terms of the metal distribution as a function of species polarity within 10min. The method was used to characterize the metal distribution in crude oils of different origins and their different fractions.

Online coupling of thin layer chromatography with matrix‐assisted laser desorption/ionization time‐of‐flight mass spectrometry: synthesis and application of a new material for the identification of carbohydrates by thin layer chromatography/matrix free material enhanced laser desorption/ionization mass spectrometry

This article describes the online hyphenation of thin layer chromatography with matrix free material enhanced laser desorption/ionization mass spectrometry (mf-MELDI-MS), the preparation of new material for MELDI and application of this newly synthesized material using TLC/MELDI-MS for the analysis of carbohydrate reference standards and plant extracts. Samples included within these analyses are standard solutions of glucose, sucrose, raffinose and a plant extract of Quercus robur, which is used for its anti-inflammatory, anti-viral and anthelminitc properties in phytomedicine. A new material for mf-MELDI-MS is prepared by immobilizing bradykinin--a peptide, on silica gel coupled to 4-(3-triethoxysilylpropylureido)azobenzene. This modification enables the absorption of laser energy sufficient for desorption and ionization of low molecular weight molecules like carbohydrates and amino acids. The newly synthesized material delivered excellent results in respect to signal-to-noise (S/N) ratio (S/N ratio: >9/1) and sensitivity (limit of detection (LOD): lower to ng/microL). Hyphenation of TLC to MELDI-MS employing the novel developed material simultaneously as chromatographic and mass spectrometric sorbent was shown for the first time for the analysis of low molecular weight molecules like mono- and oligosaccharides.

Direct analysis of Salvia divinorum leaves for salvinorin A by thin layer chromatography and desorption electrospray ionization multi‐stage tandem mass spectrometry

Salvia divinorum is widely cultivated in the US, Mexico, Central and South America and Europe and is consumed for its ability to produce hallucinogenic effects similar to those of other scheduled hallucinogenic drugs, such as LSD. Salvinorin A (SA), a kappa opiod receptor agonist and psychoactive constituent, is found primarily in the leaves and to a lesser extent in the stems of the plant. Herein, the analysis of intact S. divinorum leaves for SA and of acetone extracts separated using thin layer chromatography (TLC) is demonstrated using desorption electrospray ionization (DESI) mass spectrometry. The detection of SA using DESI in the positive ion mode is characterized by several ions associated with the compound - [M+H](+), [M+NH(4)](+), [M+Na](+), [2M+NH(4)](+), and [2M+Na](+). Confirmation of the identity of these ions is provided through exact mass measurements using a time-of-flight (ToF) mass spectrometer. The presence of SA in the leaves was confirmed by multi-stage tandem mass spectrometry (MS(n)) of the [M+H](+) ion using a linear ion trap mass spectrometer. Direct analysis of the leaves revealed several species of salvinorin in addition to SA as confirmed by MS(n), including salvinorin B, C, D/E, and divinatorin B. Further, the results from DESI imaging of a TLC separation of a commercial leaf extract and an acetone extract of S. divinorum leaves were in concordance with the TLC/DESI-MS results of an authentic salvinorin A standard. The present study provides an example of both the direct analysis of intact plant materials for screening illicit substances and the coupling of TLC and DESI-MS as a simple method for the examination of natural products.

Sphingolipidomics: a valuable tool for understanding the roles of sphingolipids in biology and disease

The sphingolipidome is the portion of the lipidome that encompasses all sphingoid bases and their derivatives. Whereas the most studied sphingoid base is sphingosine [(2S,3R,4E)-2-aminooctadecene-1,3-diol], mammals have dozens of structural variants, and hundreds of additional types have been found in other eukaryotic organisms and some bacteria and viruses. Multiplying these figures by the N-acyl-derivatives ("ceramides") and the more than 500 phospho- and glyco- headgroups places the number of discrete molecular species in the tens of thousands or higher. Structure-specific, quantitative information about a growing fraction of the sphingolipidome can now be obtained using various types of chromatography coupled with tandem mass spectrometry, and application of these methods is producing many surprises regarding sphingolipid structure, metabolism, and function. Such large data sets can be difficult to interpret, but the development of tools that display results from genomic and lipidomic studies in a pathway relational, nodal, context can make it easier for investigators to deal with this complexity.

LC-MS-based method for the qualitative and quantitative analysis of complex lipid mixtures

A simple and robust LC-MS-based methodology for the investigation of lipid mixtures is described, and its application to the analysis of human lipoprotein-associated lipids is demonstrated. After an optional initial fractionation on Silica 60, normal-phase HPLC-MS on a YMC PVA-Sil column is used first for class separation, followed by reversed-phase LC-MS or LC-tandem mass spectrometry using an Atlantis dC18 capillary column, and/or nanospray MS, to fully characterize the individual lipids. The methodology is applied here for the analysis of human apolipoprotein B-associated lipids. This approach allows for the determination of even low percentages of lipids of each molecular species and showed clear differences between lipids associated with apolipoprotein B-100-LDL isolated from a normal individual and those associated with a truncated version, apolipoprotein B-67-containing lipoproteins, isolated from a homozygote patient with familial hypobetalipoproteinemia. The methods described should be easily adaptable to most modern MS instrumentation.

Introduction to time-of-flight secondary ion mass spectrometry application in chromatographic analysis

New on-line analytical system coupling thin layer chromatography (TLC) and high selective identification unit—time of flight secondary ion mass spectrometry (TOF–SIMS) is introduced in this article. Chromatographic mixture separation and analyte surface deposition followed with surface TOF–SIMS analysis on-line allows to identify the analytes at trace and ultratrace levels. The selected analytes with different detectability and identification possibility were analysed in this hyphenated unit (Methyl Red indicator, Terpinolen and Giberrelic acid). Here, the chromatographic thin layer plays a universal role: separation unit, analyte depositing surface and TOF–SIMS interface, finally. Two depositing substrates and TOF–SIMS compatible interfaces were tested in above-mentioned interfacing unit: modified aluminium backed chromatographic thin layer and monolithic silica thin layer. The sets of positive and negative ions TOF–SIMS spectra obtained from different SIMS modes of analysis were used for analyte identification purposes. SIMS enables analyte detection with high mass resolution at the concentration level that is not achieved by other methods.

Qualitative and Quantitative Analysis by Hyphenated (HP)TLC‐FTIR Technique

In recent years, much effort has been devoted to the coupling of thin layer chromatography (TLC) and high performance thin layer chromatography (HPTLC) with spectrometric methods because of the robustness and simplicity of use of (HP)TLC and the need for detection techniques that provide identification and determination of sample constituents. Infrared (IR) is one of the spectroscopic methods that have been coupled with (HP)TLC. IR spectroscopy has a high potential for the elucidation of molecular structures; the characteristic absorption bands can be used for compound specific detection. The (HP)TLC‐FTIR coupled method has been widely used in modern laboratories for qualitative and quantitative analysis. The potential and power of this method is demonstrated by its application in various fields of analysis, such as drug analysis, forensic analysis, food analysis, environmental analysis, biological analysis, etc. The hyphenated (HP)TLC‐FTIR technique will continue to be developed in the future with the aim of taking full advantage of this method's capabilities.