Reversed-phase Thin-layer Chromatography Plates Research Articles

Direct extraction of urinary analytes from undeveloped reversed-phase thin layer chromatography plates using a solvent gradient combined with on-line electrospray ionisation ion mobility-mass spectrometry

The direct extraction of urinary analytes deposited on reversed-phase thin-layer chromatography (RP-TLC) plates is demonstrated using a solvent gradient extraction procedure without prior chromatographic development. The surface sample probe TLC-MS interface used for the gradient extraction is compared to direct loop injection into the electrospray ion source for biofluid profiling. The gradient elution is shown to enhance ion intensities, as urinary salts are eluted in aqueous formic acid in the early part of the gradient reducing ion suppression. The retention of urinary components on the C18 RP-TLC plate was confirmed by monitoring analyte responses with, and without, an aqueous wash phase prior to the solvent gradient extraction. The use of gradient elution allows fractionation of the complex biological matrix as a result of differential retention of urine components on the undeveloped RP-TLC plate. The direct gradient analysis of TLC plates has also been combined with ion mobility-mass spectrometry to further resolve the complex urinary profile and identify co-eluting compounds.

Direct analysis of pharmaceutical formulations from non‐bonded reversed‐phase thin‐layer chromatography plates by desorption electrospray ionisation ion mobility mass spectrometry

The direct analysis of pharmaceutical formulations and active ingredients from non-bonded reversed-phase thin layer chromatography (RP-TLC) plates by desorption electrospray ionisation (DESI) combined with ion mobility mass spectrometry (IM-MS) is reported. The analysis of formulations containing analgesic (paracetamol), decongestant (ephedrine), opiate (codeine) and stimulant (caffeine) active pharmaceutical ingredients is described, with and without chromatographic development to separate the active ingredients from the excipient formulation. Selectivity was enhanced by combining ion mobility and mass spectrometry to characterise the desorbed gas-phase analyte ions on the basis of mass-to-charge ratio (m/z) and gas-phase ion mobility (drift time). The solvent composition of the DESI spray using a step gradient was varied to optimise the desorption of active pharmaceutical ingredients from the RP-TLC plates. The combined RP-TLC/DESI-IM-MS approach has potential as a rapid and selective technique for pharmaceutical analysis by orthogonal gas-phase electrophoretic and mass-to-charge separation.

Rapid detection of methicillin-resistant Staphylococcus aureus by mecA gene targeted colloid gold nanoparticies probes

Objective To establish hybridization method by using the DNA-modified colloid gold nanopartieles probes for rapid and specified detection of methicillin resistant Staphylococcus aureus (MRSA). Methods DNA-modified nanoparticles probes were prepared by using two mereapto-modified mecA gene-specific oligonucleotide probes bounding with 6Ohm-diameter colloid gold nanoparticles through covalent binding. Genomic DNA of Staphylococcus aureus strains were extracted and then fragmented by ultrasonic waves. The fragmentized DNA was hybridized with the DNA-modified colloid gold nanoparticles probes. The reaction products were centrifuged and then detected by reversed-phase thinlayer chromatography plate to observe any colloid gold nanoparticles precipitation. The results of mecA gene detected by the colloid gold nanoparticles probes hybridization were compared with the results of PCR and the accuracy of the hybridization method was evaluated. Results Of total 95 tested strains, 71 strains were confirmed as MRSA and 24 swains were confirmed as methicillin susceptible Staphylococcus aureus (MSSA) by PCR. Of 71 MRSA strains, 69 strains were positive by colloid gold nanoparticles probes hybridization, the sensitivity of this method was 97.2%. All of the 24 MSSA strains were negative by using this technique. The specificity of this method was 100%. Of total 95 test strains ,93 strains were detected correctly. The accuracy was 97.9%. Conclusions Colloid gold nanoparticles probes hybridization test is well consistent with the gold standard method of PCR in detection of MRSA. Detection of MRSA by using the technique of the DNA-modified colloid gold nanopartichs probes hybridization is rapid, simple and accurate. It is potent to be a new method for rapid diagnosis of MRSA infection. Key words: MethiciIlin resistance; Staphylococcus aureus; DNA probes; Nanoparticles; Gold colloid

Isothermal DNA Amplification Coupled with DNA Nanosphere-Based Colorimetric Detection

We present a simple, rapid method for detecting short DNA sequences that combines a novel isothermal amplification method (EXPAR) with visual, colorimetric readout based on aggregation of DNA-functionalized gold nanospheres. The reaction is initiated by a trigger oligonucleotide, synthetic in nature for this proof-of-principle study, which is exponentially amplified at 55 degrees C and converted to a universal reporter oligonucleotide capable of bridging two sets of DNA-functionalized gold nanospheres. This reaction provides >10(6)-fold amplification/conversion in under 5 min. When combined with a solution containing DNA nanospheres, the bridging reporter causes nanosphere aggregation. The resulting color change from red to dark purple or blue is enhanced through spotting the solution onto a C18 reversed-phase thin-layer chromatography plate. The reaction can easily be adapted for detection of different trigger oligonucleotides using the same set of DNA nanospheres. It permits detection of as low as 100 fM trigger oligonucleotide in under 10 min total assay time, with minimal reagent consumption and requirement for instrumentation. We expect that combining this simple, versatile assay with trigger generation from a genomic target DNA sequence of interest will be a powerful tool in the development of rapid and simple point-of-care molecular diagnostic applications.

Self‐aspirating atmospheric pressure chemical ionization source for direct sampling of analytes on surfaces and in liquid solutions

A self-aspirating heated nebulizer probe is described and demonstrated for use in the direct analysis of analytes on surfaces and in liquid samples by atmospheric pressure chemical ionization (APCI) mass spectrometry. Functionality and performance of the probe as a self-aspirating APCI source is demonstrated using reserpine and progesterone as test compounds. The utility of the probe to sample analytes directly from surfaces was demonstrated first by scanning development lanes of a reversed-phase thin-layer chromatography plate in which a three-component dye mixture, viz., Fat Red 7B, Solvent Green 3, and Solvent Blue 35, was spotted and the components were separated. Development lanes were scanned by the sampling probe operated under computer control (x, y plane) while full-scan mass spectra were recorded using a quadrupole ion trap mass spectrometer. In addition, the ability to sample the surface of pharmaceutical tablets (viz., Extra Strength Tylenol and Evista tablets) and to detect the active ingredients (acetaminophen and raloxifene, respectively) selectively was demonstrated using tandem mass spectrometry (MS/MS). Finally, the capability to sample analyte solutions from the wells of a 384-well microtiter plate and to perform quantitative analyses using MS/MS detection was illustrated with cotinine standards spiked with cotinine-d3 as an internal standard.

Separation of Ionic Rhodium and Iridium Species by Electrophoresis on Reversed-Phase Thin-Layer Chromatography Plates

Reversed-phase thin-layer chromatography plates can be effectively used for the separation of metal ions in low concentrations by classical low-voltage electrophoresis. Electropherograms of the ionic composition of hydrochloric and perchloric acid solutions of rhodium(III) and iridium(IV) were obtained. These electropherograms are comparable with the results of capillary electrophoresis. Special features of electrophoresis on reversed-phase plates were noted.

Immunoaffinity column clean-up and thin layer chromatography for determination of ochratoxin A in green coffee

An immunoaffinity clean-up-based method for determining ochratoxin A (OTA) in green coffee aiming at one-dimensional thin layer chromatography (TLC) analysis was established. OTA was extracted with a mixture of methanol and aqueous sodium hydrogen carbonate solution, purified through an immunoaffinity column, separated on normal or reversed-phase (RP) TLC plates and detected and quantified by visual and densitometric analysis. The linear equation of the standard calibration curve by densitometric analysis gave R2 > 0.999 (0.04–84 ng). The mean recovery (R) of OTA from spiked samples (1.8–109 µg kg−1) by densitometric and visual analyses were 98.4 and 103.8%, respectively. The relative standard deviations (RSD) for densitometric and visual analysis varied from 1.1 to 24.9% and from 0.0 to 18.8%, respectively. The RSD for naturally contaminated samples by densitometry (three levels of contamination, n = 3) varied from 11.1 to 18.1%. The correlation (R2) between high-performance liquid chromatography (HPLC) and densitometry, and between visual and densitometric analysis for spiked samples were > 0.99. The limit of detection (LOD) of the method was 0.5 µg kg−1 for normal TLC. Toluene-ethyl acetate-88% formic acid (6:3:1 v/v/v) and acetonitrile-methanol-water-glacial acetic acid (35:35:29:10 v/v/v/v) were regarded as the suitable TLC solvents for eluting both standards and samples on normal and RP TLC plates, respectively. Toluene-acetic acid (99:1 v/v) was chosen as the spotting solvent among several others for giving the best sensitivity and resolution of OTA on TLC plates as well as the best recovery of OTA from standard and sample extract residues. Preliminary studies were carried out to investigate the reuse of the immunoaffinity column and the interference of caffeine in the OTA recovery.

Electrofilament deposition and off-column detection of analytes separated by capillary electrophoresis.

Capillary electrophoresis interfaced with electrospray is a convenient technique for continuously transferring column effluent from capillary-to-planar format. Conditions are optimized to produce a narrow (approximately 20 microm) liquid filament (electrofilament), which is capable of depositing spatially focused bands with track widths that are routinely 100 microm. A fiber optic-based, laser-induced fluorescence cell is employed to monitor the separation on-column while the separated bands are deposited onto a moving substrate. The photodetection of deposited bands is accomplished by using either a charge-coupled device camera or a photomultiplier tube. Deterioration of on-column separation performance is observed when the electrofilament voltage is applied. Elevating the inlet of the capillary column, to provide hydrodynamic flow, restores separation performance. Substrate temperature and translational rates are optimized with respect to both off-column separation efficiency and signal intensity. Off-column separation efficiencies of 65 000 plates per meter were achieved. A linear dynamic range of 10(3) and a limit of detection of 10(-8) M were obtained for kiton red deposited onto a reversed phase thin-layer chromatography plate. To demonstrate the applicability of this technique to more complex separation solutions, a dye mixture was successfully separated and deposited with sodium dodecyl sulfate in the running buffer.

Effect of β-cyclodextrin derivatives on the retention of steroidal drugs

The retention of eighteen steroid drugs was determined on a β-cyclodextrin polymer (βCDP)-coated silica column using methanol-water mixtures as eluents. The relative strength in inclusion formation between the drugs and hydroxypropyl-βCD (HPβCD) and dimethyl-βCD was determined by charge transfer chromatography carried out on reversed-phase thin-layer chromatography plates. The retention characteristics of drugs were correlated with their physicochemical parameters and with their inclusion complex-forming capacity. Calculations indicated that the inclusion complex-forming capacity of the drugs has little impact on the retention that is due to the HPβCD and water-insoluble βCD polymers exposed to different retention characteristics. The hydrophilic molecular parameters of drugs significantly influenced their retention. This result suggests that the selectivity of the βCDP-coated column may be different from that of the traditional reversed-phase columns.

Analysis of Metribuzin and Associated Metabolites in Soil and Water Samples by Solid Phase Extraction and Reversed Phase Thin Layer Chromatography

Abstract A method to analyze metribuzin, 4-amino-6-(1,1-dimethylethyl)-3-(methylthio)-1,2,4-triazin-5(4H)-one, and its major metabolites, deaminated metribuzin, DA, 6-(1,1-dimethylethyl)-3-(methylthio) -1,2,4-triazin-5(4H)-one, diketometribuzin, DK, 4-amino-6-(1,1-dimethylethyl)-1,2,4-triazin-3,5(2H,4H) -dione and deaminated diketometribuzin, DADK, 6-(1,1-dimethylethyl)-1,2,4-triazin-3,5(2H,4H)-dione in soil and water samples by reversed-phase thin layer chromatography (RPTLC) is described. Soil samples were extracted with MeOH:0.01 M CaCl2.2H2O (4:1) and the extracts filtered to remove the sediment. The extract then was diluted with deionized water and eluted through a C18, solid phase extraction (SPE) cartridge. Water samples (150-mL) were eluted directly through C18, SPE cartridges. Both soil and water extracts were chroma-tographed on pre-coated RPTLC plates. The concentration of metribuzin was determined by UV densitometry at 290 nm. Recoveries of metribuzin from soil samples fortified at 2 mg kg−1 r...

Comparison of non-hydrophobic C 18 bonded and paraffin-coated silica gel reversed-phase thin-layer chromatography plates

The chromatographic properties of C 18 bonded reversed-phase thin-layer chromatography plates have been compared with paraffin-coated silica gel thin-layer chromatography plates for the reversed-phase chromatography of a range of organic compounds. These included non-steroidal anti-inflammatory drugs, ecdysteroids, aminophenols, hydroxybenzoic acids and antipyrine. Both types of plate can be used with mobile phases containing up to 100% water, allowing a wide choice of solvent systems. Although differences were found between bonded and coated plates in most applications they are interchangeable following minor adjustments to the mobile phase.

Choice of methods for the determination of abscisic acid in plant tissue

Two standard procedures are described for the determination of abscisic acid in plant tissue. One includes partial but rapid clean-up followed by semi-preparative high-performance liquid chromatography on a 5-μm reversed-phase C 18 column in preparation for quantitative analysis by electron-capture gas—liquid chromatography (GLC). The alternative method employs Clin Elut tubes plus pre-adsorbent and reversed-phase thin-layer chromatography plates. The need for freezing precipitation, separating funnels, polyvinylpyrrolidone slurries or glass extraction columns has been eliminated from both methods. Improved resolution during GLC has been achieved by using a column packing with an ultra-thin phase of Carbowax 20M and by careful choice of the injection solvent. In addition to electron-capture detection and UV isomerization, further confirmation of identity is obtained by flame-ionization detection after injection of the sample into a fused-silica capillary column.

Reversed-phase thin-layer chromatography for the separation and analysis of ecdysteroids

Reversed-phase thin-layer chromatography plates of different types and different manufacture are all suitable for the chromatography of ecdysteroids (insect moulting hormones). A number of solvent systems have been examined, and eluation with methanol-water mixtures from C 18-coated plates provides a suitable general system. The detection limit of ecdysteroids on reversed-phase plates is 10- -7 g, using fluorescence quenching or vanillin-sulphuric acid spray. The recovery of ecdysteroids, which is inefficient from noram silica plates, is much more efficient from reversed-phase plates, especially when residual silinol groups are “capped” with organosilane reagents.