Quantitative measurements on wetted thin layer chromatography plates using a charge coupled device camera

This paper presents the first study of imaging of spots on thin-layer chromatographic plates whilst still wet with solvent. Imaging and quantification of Sudan II after development with dichloromethane was carried out in both reflectance and transmission modes, using a charge coupled device (CCD) camera. The relationship between peak area and sample loading was established at low sample loading, and found to be linear over an order of magnitude for both wet and dry modes with r2-values > 0.99. All data processing was carried out using the Beer-Lambert equation. Curvature at high loadings in the plots of integrated absorbance as a function of sample loading was accounted for using an empirical expression designed for use with the Kubelka-Munk treatment and apparent absorbance of the stationary phase due to scattering. Results are consistent with an effective pathlength significantly longer than the thickness of the sorbent layer. The limit of detection on a dry plate (0.5 ng) was found to be lower than on a wetted plate (2 ng). Precision was found to be 1-4% RSD intra-plate and 8-14% RSD inter-plate. Results are compared with quantification of the same analyte on dried plates.     

Development and validation of a simple thin layer chromatographic method for the analysis of artemisinin in Artemisia annua L. plant extracts

Owing to the development of parasite resistance to standard antimalarial treatments like chloroquine and sulfadoxine-pyrimethamine, the demand for Artemisia annua, a key ingredient for new and highly effective antimalarial drugs, is huge. Therefore selective and precise methods to determine the content of artemisinin in dry plant material and in raw impure extracts are needed. In this work a method is described for the clear separation and extraction of artemisinin from other plant components in the Artemisia annua L. plant by thin-layer chromatography (TLC). To obtain optimal extraction and recovery efficiency, several parameters were evaluated, including choice of extraction solvent, TLC plate type and sensitivity between UV and visible light. Method validation was performed on both the dry plant material and non-purified plant extracts. Toluene presented the highest extraction efficiency compared with petroleum ether, hexane and methanol. Reversed-phase plates showed more concentrated spots than normal-phase plates, while the sensitivity of the analysis in UV was comparable to that in visible light but less precise. The impure plant extracts were analyzed by both TLC and HPLC-UV at 215 nm and both methods met the requirements for linearity, selectivity, precision and accuracy. Hence, the proposed TLC method can easily be used for both qualitative and quantitative control of the raw plant extract in areas where advanced methods are scarce.     

HPTLC densitometric analysis of arbutin in bulk drug and methanolic extracts of Arctostaphylos uva-ursi

A high-performance thin layer chromatographic densitometric method for the analysis of arbutin was developed and validated in the present investigation. Arbutin was separated on aluminium-backed silica gel 60 F(254) plates with methanol?:?chloroform (3:7)% (v/v) as the mobile phase. This system was found to give a compact spot of arbutin at a retention factor (R(f)) value of 0.32 ± 0.02. The limit of detection and limit of quantification were found to be 35.42 and 106.26 ng/spot, respectively. The proposed method with a high degree of precision and accuracy was employed for the analysis of arbutin in the bulk drug and methanolic extract of Arctostaphylos uva-ursi.     

Evaluation of a method for high throughput solubility determination using a multi-wavelength UV plate reader

Aqueous solubility is a critical physicochemical property and must be addressed early during drug discovery research. Due to the difficulty in accurately predicting aqueous solubility in silico, high throughput experimental determination of aqueous solubility is in great demand. This study evaluates a method using a multi-wavelength UV plate reader and disposable 96-well UV plates for fast solubility determination. It was demonstrated that this method has the sensitivity and reproducibility to effectively determine solubility as low as 1 micro M. Excellent correlation (R>0.97) was observed between the solubility determined using the UV reader method and the HPLC method over the range of 1-1000 micro M for a diverse set of pharmaceutical compounds. In addition to excellent sensitivity and reproducibility, the UV plate reader method also offers the flexibility of being able to determine thermodynamic solubility in the presence or absence of dimethyl sulfoxide, which is a solvent widely used for combinatorial compounds during high throughput screening.     

A new HPTLC densitometric method for analysis of swertiamarin in Enicostemma littorale and commercial formulations

A high-performance thin layer chromatographic densitometric method for the analysis of swertiamarin in 60% methanolic extract of Enicostemma littorale and commercial formulations has been developed and validated in this study. Swertiamarin was separated on aluminium-backed silica gel 60 F??? plates using ethyl acetate : methanol : water (77 : 15 : 8)% v/v as the mobile phase. This system was found to give a compact spot of swertiamarin at R(f) value 0.36 ± 0.01. The limit of detection and limit of quantification were found to be 31.25 and 103.12 ng spot?1, respectively. The proposed method was employed with a high degree of precision and accuracy for the estimation of swertiamarin in methanolic extract of Enicostemma littorale and in commercial formulations.     

Dissolution assay of theophylline, diprophylline and proxyphylline from a sustained release dosage form by high performance thin layer chromatography.

The content and dissolution rate of theophylline, diprophylline and proxyphylline from a sustained release formulation were determined by UV in situ densitometry. After separation the chromatographic zones corresponding to the spots of theophylline, diprophylline and proxyphylline on the high performance thin layer chromatographic plates were scanned in reflectance/absorbance mode at 275 nm. Quantification was performed with a second degree polynomial function over the range 40-200 ng for theophylline and 60-300 ng for diprophylline and proxyphylline. Percentages of dissolved theophylline, diprophylline and proxyphylline were monitored over 1, 3 and 6 h. The method was found to be simple, accurate, reliable, time-saving (up to 18 samples can be determined simultaneously) and low-cost.     

Determination of amitriptyline and nortriptyline in human plasma by quantitative thin-layer chromatography

A thin-layer chromatographic method for simultaneous determination of amitriptyline (AT) and nortriptyline (NT) in human plasma is described. Both substances are extracted from biological material by means of a single extraction. The extract is evaporated until dry and the residue quantitatively applied to a silica gel thin-layer plate. AT and NT are separated from interfering plasma components by chromatography. The spots are visualized by nitration, reduction and coupling with N-(1-naphtyl)ethylenediamine on the plate. The intensity of the azo-dyes formed can be measured densitometrically. Using 1 ml of plasma, the sensitivity limit was 0.5 ng/ml for both substances. About 10--15 plasma samples can be analysed per day. The method is applicable to pharmacokinetic studies after a single oral dose of 25 mg AT as hydrochloride in man.     

Normal Raman and surface enhanced Raman spectroscopic experiments with thin layer chromatography spots of essential amino acids using different laser excitation sources

A comparative study of the feasibility and efficiency of Raman spectroscopic detection of thin layer chromatography (TLC) spots of some weak Raman scatterers (essential amino acids, namely, glycine and L-forms of alanine, serine, valine, proline, hydroxyproline, and phenylalanine) was carried out using four different visible and near-infrared (NIR) laser radiations with wavelengths of 532, 633, 785, and 1064 nm. Three types of commercial TLC plates were tested and the possibility of inducing surface enhanced Raman scattering (SERS) by means of Ag-sol was also investigated. The spectra obtained from spotted analytes adsorbed on TLC plates were of very different quality strongly depending on the excitation wavelength, the wetness of the samples, and the compounds examined. The best results were obtained with the simple silica TLC plate, and it has been established that the longest wavelength (lowest energy) NIR excitation of a Nd:YAG laser is definitely more suitable for generating normal Raman scattering of analyte spots than any of the visible radiations. Concerning SERS with application of Ag-sol to the TLC spots, 1-3 orders of magnitude enhancement was observed with wet samples, the greatest with the 532 nm radiation and gradually smaller with the longer wavelength excitations. It is shown, however, that due to severe adsorption-induced spectral distortions and increased sensitivity to microscopic inhomogeneity of the sample, none of the SERS spectra obtained with the dispersive Raman microscope operating in the visible region were superior to the best NIR normal FT-Raman spectra, as far as sample identification is concerned.     

Application of iodine-azide reaction for detection of amino acids in thin-layer chromatography

The iodine-azide reaction was employed to TLC detection of sulphur-containing derivatives of protein and some non-protein amino acids. The derivatization reaction with phenyl isothiocyanate (PITC) took place directly on the plate before the developing step. Subsequently, the plates were sprayed with a mixture of sodium azide and starch solution in NP-TLC and in the case of RP-TLC sodium azide solution with starch incorporated into mobile phase and then exposed to iodine vapour. The spots became visible as white spots on violet-grey background. The obtained detection limits of PTC-derivatives have been compared with other visualizing techniques commonly used in TLC practice (UV254 and iodine vapour). The iodine-azide system has been proved to be the most favourable and enabled to detect quantities per spot in the range of 1-60 pmol (HPTLC) and 3-100 pmol (TLC).     

UV radiation effects on plant growth and forage quality in a shortgrass steppe ecosystems

Levels of UV were manipulated in a native shortgrass steppe using open-sided structures with tops that either passed or blocked wavelengths shorter than approximately 370 nm. Precipitation was controlled to create a drought or a very wet year. Subplots were either nondefoliated or defoliated to simulate grazing by livestock, which is the primary land use. Plant community productivity and forage quality were assessed in response to the two climate change variables (UV, precipitation) and grazing stress. Productivity and seasonal standing biomass of the dominant grass species were negatively affected by passing versus blocking UV, but only in the dry year. Another species was negatively affected by passing UV in the wet year, indicating the potential for future shifts in species composition. Forage quality for ruminants increased when UV was passed compared with blocked, as determined by in vitro digestible dry matter, depending on species and precipitation. Nitrogen concentrations and soluble and fiber components of vegetation also displayed some UV effects, but they were generally small and depended on species, season or amount of precipitation (or all). Grazing treatment had large positive effects on current-year productivity only in the wet year and some small positive effects on quality in both wet and dry years. Interactions between UV and grazing treatment were not observed.     

Quantitative determination of drugs by in situ spectrophotometry of chromatograms for pharmacokinetic studies. I. Sulpiride and other benzamides, vincamine, naftazone (author's transl)

Assays are proposed for sulpiride and other benzamides, vincamine and naftazone in plasma (or blood) and urine with direct UV reflectance spectrophotometry on this are applied directly on TLC along with a calibration curve on each plate. Plasma (or total blood) samples are extracted, and an internal standard is added before aplication; slopes of the obtained calibration curves do not change significantly from plate to plate, thus allowing several determinations on the same plate. The sensitivity is 2 microgram in a 1-ml sample (amount applied 30 ng) for sulpiride and related compounds and about the same for vincamine. Naftazone is determined in plasma with simultaneous reflectance and transmittance spectrophotometric measurements at 520 nm on chromatoplates sprayed with lead acetate, the sensitivity reached is 10 ng in a 1-ml sample (amount applied 0.5 ng). For all drugs studied, the proposed techniques are specific, reliable and sensitive enough and can be used to perform pharmacokinetic studies in human or in animal after administration of doses in the therapeutic range.     

On-Plate Electrochemical Detection for Thin-Layer Chromatography

Abstract

Voltammetric electrochemical detection (ECD) coupled with thin-layer chromatography (TLC) was demonstrated for the quantification of trace organic compounds directly on a TLC plate. the electrochemical detection solvent was a thin layer of aqueous potassium chloride. For undeveloped plates, detection limits for p-anisidine and p-phenetidine were 10 ng and 13 ng, respectively. Linearity was demonstrated over nearly 2 orders of magnitude. After development, detection limits increased by approximately a factor of ten. Advantages of this method over other quantitative TLC methods include sensitivity, speed, simplicity, and cost. in addition, this method is selective for electrochemically active compounds. Major sources of experimental error include spot size reproducibility, working electrode placement, and supporting electrolyte film thickness.     

Stability-Indicating TLC Method for the Determination of Dutasteride in Pharmaceutical Dosage Forms

A simple, sensitive, selective, precise and stability-indicating thin-layer chromatographic method for determination of dutasteride both as a bulk drug and as pharmaceutical tablets was developed and validated as per the International Conference on Harmonization guidelines. The method employed thin-layer chromatography aluminium plates precoated with silica gel 60F₂₅₄ as the stationary phase and the mobile phase consisted of acetonitrile:methanol:dichloromethane in the ratio of 2.0:1.0:2.0, v/v/v. This solvent system was found to give compact spots for dutasteride (R _f value of 0.64 ± 0.02). Densitometric analysis of dutasteride was carried out in the absorbance mode at 244 nm. The linear regression analysis data for the calibration plots showed good linear relationship with r = 0.9943 with respect to peak area in the concentration range of 100–600 ng per band. The method was validated for precision, accuracy, ruggedness and recovery. The limits of detection and quantitation were 7.54 and 22.85 ng per band, respectively. Dutasteride was subjected to acid and alkali hydrolysis, oxidation, photo degradation, dry heat and wet heat treatment. The drug undergoes degradation under acidic, basic conditions, photolytic, oxidative and upon wet and dry heat treatment. The degraded products were well separated from the pure drug. The statistical analysis proves that the developed method for quantification of dutasteride as bulk drug and from pharmaceutical tablets is reproducible and selective. As the method could effectively separate the drug from its degradation products, it can be employed as stability-indicating.     

Thin layer chromatography–spray mass spectrometry: a method for easy identification of synthesis products and UV filters from TLC aluminum foils

A straightforward procedure for direct mass spectrometric (MS) analysis of spots from thin layer chromatography (TLC) plates, without the need of an external ion source, was developed using the aluminum plate backing as spray tip. The spots were cut out shaped as a tip with a 60° angle, mounted in front of the MS orifice, and after addition of a spray solvent spectra were obtained immediately. A high-resolution time-of-flight MS was used since the method is of particular interest for rapid identification or confirmation of spots from TLC plates. The practical benefits of this technique were demonstrated by detection of by-products of organic reactions, by identification of degradation products, and by accurate confirmation of spots when UV filters in sunscreens were analyzed by TLC. Employing the described method TLC spots can be evaluated fast without the need of an external ion source or devices for analyte transfer from TLC to MS, only a basic MS instrument and a high-voltage power supply is required.

Derivatisation of sympathomimetics with primary amino groups on thin-layer plates by spotting the sample spots with fluorescamine

Summary A method has been developed for the quantitative determination of sympathomimetics containing a primary amino group on thin-layer plates with fluorescamine. The reaction is carried out by spotting fluorescamine solution in acetone on top of the sample spots. The fluorescamine derivatives are subsequently separated using appropriate solvent systems. Spotting a buffer before reaction and spraying with triethanolamine after development is unnecessary. The consumption of reagent is extremely low. For ten thin-layer plates with ten sample spots per plate only 0.2 cm³ reagent solution are needed. The method has been applied to the quantitative determination of some compounds with primary amino groups in pharmaceutical preparations.Dedicated to Prof. Dr. G. Zigeuner on his 60^th birthday.     

Evaluation of poly(ethylene terephthalate) photostabilisation using FTIR spectrometry of evolved carbon dioxide

Carbon dioxide evolution from poly(ethylene terephthalate) (PET) films during ultraviolet (UV) exposure has been monitored using FTIR interrogation of the atmosphere surrounding the test pieces. Measurement periods as little as 4 h could easily discriminate between CO₂ emission rates when tests were conducted to investigate the effect of using different reaction atmospheres or of including UV absorber in the PET samples. Samples containing UV absorbers either homogeneously distributed through the film or in thin surface layers (∼0.7 μm thick) were also tested. Relatively small reductions in CO₂ emission rates were observed with samples containing UV absorbers but the rates were not very sensitive to the distribution and concentration of the absorbers. A thin surface layer containing only 2% stabiliser (equivalent to 0.23% stabiliser when averaged over the whole film thickness) provided oxidation reduction similar to that observed when 1% stabiliser was distributed evenly throughout the sample. Tests were conducted in wet oxygen, dry oxygen and dry nitrogen. For as-received bi-axially drawn PET film containing no absorber, the CO₂ emission rate under UV illumination in wet oxygen was much higher than in dry oxygen or dry nitrogen. For as-received PET the difference between the rates observed in dry oxygen and dry nitrogen was small. For PET films that had been pre-exposed to UV (for 9 days) prior to insertion into the in situ CO₂ measurement cell the rate of CO₂ generation in oxygen was significantly larger than that in nitrogen. In both nitrogen and oxygen the presence of UV absorbers significantly decreased the rate of CO₂ generation.     

Capillary electrochromatography of complex plasma matrix on a C18/SCX column using UV-vis and mass spectrometric detection

Summary Separations of compounds in plasma were performed on a Hypersil Duet C₁₈/SCX capillary electrochromatography (CEC) column, utilizing an automated injection system combined with a short in-house designed and fabricated micro-electrospray CEC mass spectrometer interface. Protein precipitation was used prior to the CEC separations. More than two hundred separations of the corticosteroids Dexamethasone and Betamethasone 17-valerate, and Fluticasone Propionate in complex plasma matrix were performed on a single column under isocratic conditions. The method demonstrated good reproducibility, selectivity, sensitivity and high efficiencies. Linear calibration with good correlation was typical. Estimated detection limits in the low micromolar and nanomolar range for all compounds were obtained using UV-Vis absorbance (UV) and Electrospray Mass Spectrometric (ES/MS) detection respectively. Efficiencies for all compounds were typically 87,500 plates on a 25 cm column (350,000 plates m⁻¹) and increased with the number of plasma samples injected, up to 250,000 plates per column (1,000,000 plates m⁻¹). These very high observed plate counts may be artificially enhanced by the inadequate scan possibilities of the MS over very narrow chromatographic peaks.     

Stability-Indicating HPTLC Method for the Determination of Tamsulosin in Pharmaceutical Dosage Forms

A simple, sensitive, selective, precise and stability indicating high-performance thin-layer chromatographic method was developed for the determination of tamsulosin (TAM) in bulk and tablet formulation. Validation was carried out in compliance with International Conference on Harmonization guidelines. The method employed thin-layer chromatography aluminium plates pre-coated with silica gel 60F₂₅₄ as the stationary phase and the mobile phase consisted of acetonitrile/methanol/dichloromethane (2.0: 1.0: 2.0, v/v/v). This solvent system was found to give compact spots for tamsulosin (R _f = 0.27 ± 0.02). Densitometric analysis of TAM was carried out in the absorbance mode at 286 nm. Linear regression analysis showed good linearity (r ² = 0.9993) with respect to peak area in the concentration range of 300–800 ng per band. The method was validated for precision, accuracy, ruggedness and recovery. Limits of detection and quantitation were 8.49 and 25.72 ng per band, respectively. TAM was subjected to acid and alkali hydrolysis, oxidation, photo degradation, dry heat and wet heat treatment. The drug underwent degradation under acidic, basic and photolytic conditions. The degraded products were well separated from the pure drug. Statistical analysis proved that the developed method, used for quantification of TAM as a bulk drug and present in pharmaceutical tablets, was reproducible and selective.     

Stability-Indicating HPTLC Method for the Determination of Tamsulosin in Pharmaceutical Dosage Forms

A simple, sensitive, selective, precise and stability indicating high-performance thin-layer chromatographic method was developed for the determination of tamsulosin (TAM) in bulk and tablet formulation. Validation was carried out in compliance with International Conference on Harmonization guidelines. The method employed thin-layer chromatography aluminium plates pre-coated with silica gel 60F₂₅₄ as the stationary phase and the mobile phase consisted of acetonitrile/methanol/dichloromethane (2.0: 1.0: 2.0, v/v/v). This solvent system was found to give compact spots for tamsulosin (R _f = 0.27 ± 0.02). Densitometric analysis of TAM was carried out in the absorbance mode at 286 nm. Linear regression analysis showed good linearity (r ² = 0.9993) with respect to peak area in the concentration range of 300–800 ng per band. The method was validated for precision, accuracy, ruggedness and recovery. Limits of detection and quantitation were 8.49 and 25.72 ng per band, respectively. TAM was subjected to acid and alkali hydrolysis, oxidation, photo degradation, dry heat and wet heat treatment. The drug underwent degradation under acidic, basic and photolytic conditions. The degraded products were well separated from the pure drug. Statistical analysis proved that the developed method, used for quantification of TAM as a bulk drug and present in pharmaceutical tablets, was reproducible and selective.

     

Further data on a histochemical reaction as applied to the thin-layer chromatography of sterols.

A method for the characterization of sterols on silica gel G layers is proposed. After the chromatograms have been sprayed with a permanganate-sulphuric acid oxidative reagent and the reaction has been terminated with sodium hydrogen sulphite, the plates are sprayed with the colour-developing reagent (an acid solution of alcian blue or toluidine blue). The plates are also viewed under UV radiation (254 and 366 nm). Only 3 of the 28 sterol samples assayed (spot content 8 mug) did not show positive reactions after oxidation, which suggests that this step can be used as a "universal" detection method for sterols. After staining the plate, several sterols are shown to be easily differentiated from one another. The exposure of the plates to UV radiation assists characterization. In general, the reaction exhibits satisfactory sensitivity for the qualitative and differentiating detection of sterols; it is also rapid and easy to carry out.