Simultaneous quantification of five marker compounds of Betula utilis stem bark using a validated high-performance thin-layer chromatography method

A sensitive, selective and robust densitometric high-performance thin-layer chromatographic method was developed and validated for five marker compounds, namely betulin, lupeol, oleanolic acid, 3-acetyloleanolic acid and β-sitosterol, known for their various therapeutic activities. The marker compounds have been isolated from the stem bark of Betula utilis, well characterized by the spectral analysis, and their simultaneous quantitative determination carried out by high-performance thin-layer chromatography (HPTLC) method. The resolution of marker compounds was carried out on silica-gel 60 plates, using n-hexane:ethyl acetate (8:2 v/v) as the mobile phase. The HPTLC densitometry was performed at 500-nm wavelength after the post chromatographic derivatization with ceric ammonium sulfate reagent. The optimized method provided good linear relation (r>0.9960) for all the investigated analytes. The method is simple, and reproducible, which may be applied for quantitative analysis of the above-mentioned marker compounds.     

Pyrimidine nucleoside phosphorylase assay by automated high-performance liquid chromatography

A new assay for pyrimidine nucleoside phosphorylase is reported. This method utilizes an isocratic reversed-phase high-performance liquid chromatographic system for separation of nucleosides and bases. Product detection is accompanied by ultraviolet monitoring and radioactive flow detection. Use of an automated sample injector allows for the analysis of a series of samples, with data recorded onto a microprocessor-based cassette recorder. Data can then be downloaded into computer memory. The velocity of uridine phosphorylase (E.C. 2.4.2.3) was a linear function of enzyme concentration. The Michaelis constant for uridine at pH 8.0 was found to be in close agreement with the value obtained by a thin-layer chromatographic assay method.     

Application of thin layer and high performance liquid chromatography to the separation and determination of cephalexin in cephradine,in bulk powder and in pharmaceuticals

Summary A thin-layer chromatographic procedure for the quantitative determination of cephalexin in cephradine formulations is described. The thin-layer chromatography is carried out on a silica gel layer and the quantitation is by UV scanning of the chromatogram. The procedure was transferred to high-performance liquid chromatography on a microparticulate silica gel column with a similar eluent system. The cephalexin content of a few commercial cephradine samples is determined by using these techniques. The results of these procedures are compared to those obtained with high-performance liquid chromatography on a C 18 reverse phase column.     

Caffeine Determination in Human Saliva and Urine by TLC and Ultraviolet Absorption Densitometry

A quantitative method using thin-layer chromatography (TLC) plates with concentrating zone and ultraviolet absorption densitometry is described for caffeine (CAF) determination in human saliva and urine. The applicability of the CAF method was tested for saliva and urine from male individuals. The changes of salivary CAF, urinary CAF concentration and of urinary CAF excretion correspond with results of previous investigations using gas chromatography, high performance liquid chromatography, radioimmunoassay or enzyme immunoassay methods. In summary, the thin-layer chromatography/densitometry method here described is easy to perform, reproducible and accurate. Thus, it is suitable for routine analysis of CAF in human saliva or urine, being an effective alternative to other, more expensive and more time-consuming chromatographic or immunological methods.     

Fluorescence determination of 5-fluorouracil and 1-(tetrahydro-2-furanyl)-5-fluorouracil in blood serum by high-performance liquid chromatography

Fluorescence derivatization of 5-fluorouracil (5-FU) and 1-(tetrahydro-2-furanyl)-5-fluorouracil (ftorafur, FT) with 4-bromomethyl-7-methoxycoumarin using 18-crown-6 as a catalyst is studied with aim of developing a sensitive and selective liquid chromatographic method. 5-FU and FT form virtually substituted derivatives which possess maxima in their fluorescence emission spectra near 400 nm. These derivatives are separated by thin-layer chromatography and high-performance liquid chromatography to confirm the completion of reaction. For the determination of 5-FU and FT in serum, the reversed-phase high-performance liquid chromatographic separation of the derivatives is studied with a C13 column. This chromatography is of importance for the accurate determination of 5-FU and FT, which are, respectively, an important antitumour agent for the treatment of solid tumours in clinical medicine and a masked form of 5-FU generated in vivo.     

Development and validation of a method for measuring the glycine and taurine conjugates of bile acids in bile by high-performance liquid chromatography

We developed and validated a simple method for measuring the individual glycine and taurine conjugates of bile acids in bile by high-performance liquid chromatography with a C18 reversed-phase column using an isocratic solvent system of acidified methanol--potassium phosphate. Without preliminary derivatization or purification, complete separation of the ten major conjugated bile acids present in bile could be achieved in 65 min. Total bile acid concentrations were identical when measured enzymatically and by summing the individual bile acids determined by high-performance liquid chromatography. Bile acid composition determined by gas-liquid chromatography correlated with results by high-performance liquid chromatography. Finally, measurements of individual glycine and taurine conjugates in human bile and in mixtures of bile acid standards by high-performance liquid chromatography and thin-layer chromatography gave similar results. This high-performance liquid chromatographic system permits simultaneous quantification of total and individual bile acids and their glycine and taurine conjugates in bile.     

Chromatographic identification of pesticides

The chromatographic properties of 51 common pesticides have been measured using seven different chromatographic systems involving gas-liquid chromatography (GLC), high-performance liquid chromatography (HPLC) with diode-array spectrophotometric detection and thin-layer chromatography (TLC) with different spray reagents. Correlation coefficients were calculated for combinations of all systems. The best combination of the chromatographic systems examined for the identification of an unknown compound is GLC on OV-17, HPLC on ODS-Hypersil with acetonitrile-water as eluent and TLC using an isooctane-ethyl acetate solvent system.     

Thin-layer chromatographic and column liquid chromatographic analyses of morphine in urine via dabsylation

A semiquantitative screening method for morphine in urine and a quantitative assay method for the drug were developed. In the semiquantitative method, morphine in urine was directly reacted with 4-dimethylaminoazobenzene-4'-sulphonyl chloride (dabsyl chloride) in a slightly alkaline medium. The orange-coloured dabsyl morphine was separated by silica gel thin-layer chromatography and the spot intensity was visually compared with that of the standards. The limit of detection is 0.075 microgram/ml. In the quantitative method, morphine was extracted from urine before dabsylation. The dabsylation reaction is very fast and is complete within 5-10 min at room temperature. Dabsylation yield is maximum at a dabsyl chloride concentration of 6.2 mM. Total recovery of morphine using the extraction and dabsylation procedures described is 66%. Dabsyl morphine, thus formed, was analysed using high-performance liquid chromatography by monitoring its absorbance at 436 nm on a normal-phase mu Porasil column. The limit of quantitation using high-performance liquid chromatography is 0.26 microM (0.075 microgram/ml), which corresponds to 10.5 pmol of injected dabsyl morphine. Quantitative assay was also carried out by thin-layer chromatography on silica gel followed by densitometry. The limit of quantitation is 1.3 microM (0.375 microgram/ml).     

Studies on neurosteroids. IX. Characterization of estrogens in rat brains using gas chromatography-tandem mass spectrometry.

The characterization of the classical estrogens (estrone, estradiol, estriol) and guaiacol estrogens (2-hydroxyestrone 3-methyl ether, 4-hydroxyestrone 3-methyl ether) in rat brains was performed using gas chromatography-tandem mass spectrometry (GC-MS-MS). Estrogens were purified from Wistar strain rat brains by some chromatographic pre-treatments, such as solid-phase extraction, preparative thin-layer chromatography or preparative high-performance liquid chromatography. After the derivatization with O-methylhydroxylamine and/or N,O-bis(trimethylsilyl)trifluoroacetamide, estrogens were identified by comparison of their chromatographic behavior during GC-MS-MS operating in the product ion scan mode and comparison with the product ion MS spectra of an authentic sample. These evidences suggested that estrogens exist in rat brains as neurosteroids or neuroactive steroids.     

Determination of aflatoxins in food products by chromatography.

Several chromatographic methods for the determination of aflatoxins in agricultural and food products are reviewed. During the past two decades, identification and determination of aflatoxins were done by thin-layer chromatography (TLC) because it was easy, fast and inexpensive. However, high-performance liquid chromatography (HPLC) using fluorescence detection is now the method of choice for determining aflatoxins and is also growing in popularity for their identification. The reasons for selecting HPLC over TLC can be summarized as the ability to analyze for a wide variety of compounds, including compounds that are easily degraded by heat, light or air, the ease of adaptation to confirmatory procedures, the potential for automation and the dramatic improvement in instrumentation, including the development of increasingly sensitive fluorescence and electrochemical detectors and short, high-resolution, reversed-phase columns.     

Isolation of palm tocols using supercritical fluid chromatography

Crude palm oil contains 600 to 1000 ppm of tocols in the form of tocopherols and tocotrienols. These palm tocols have been isolated and analyzed in the past by various chromatographic techniques such as open column chromatography, high-performance liquid chromatography, as well as thin-layer chromatography. Supercritical fluid chromatography (SFC) has emerged as a more advanced chromatographic technique in recent years. The tocols present in palm oil are successfully isolated using SFC. Identification of these tocols is supported by various spectroscopic techniques such as 1H NMR, 13C NMR, and mass spectrometry.     

HPTLC analysis of organotin compounds in preservative solutions and preservative-treated wood

A high-performance thin-layer chromatographic (HPTLC) method is described for the determination of tributyltin compounds (bis(tri-n-butyltin) oxide, TBTO, and tri-n-butyltin naphthenate, TBTN) and their degradation products (dibutyltin and monobutyltin compounds). The organotin compounds are extracted from wood with ethanol containing 0.5% (v/v) of hydrochloric acid and the separation of the defferent kinds of organotin compounds is achieved by thin-layer chromatography. The sample spots are measured using a scanning densitometer after decomposing the organotin compounds to inorganic tin by ultraviolet irradiation and visualization of the spots with pyrocatechol violet. Applications of the method to detection and quantification of organotin compounds in preservative solutions, in recently impregnated wood, and in wood samples from five-year-old window frames are described.     

Determination of streptomycin in serum by high-performance liquid chromatography

A high-performance liquid chromatographic method was developed for monitoring the serum concentration of streptomycin. The method includes clean-up using a Sep-Pak C18 cartridge and quantitation using dihydrostreptomycin as an internal standard. Streptomycin and dihydrostreptomycin were separated by reversed-phase ion-pair chromatography on LiChrosorb RP-18 and detected by UV absorption (195 nm). The calibration graph of serum streptomycin concentration was linear over the range 5-50 micrograms/ml. Streptomycin was added to serum at the level of 20.0 micrograms/ml and its concentration was determined to be 18.9 micrograms/ml with a coefficient of variation of 2.07% (n = 5). The clinical application of this method was confirmed by comparison with fluorescence polarization immunoassay.     

Planar chromatographic method development using the PRISMA optimization system and flow charts

This study presents a modern planar chromatographic method-development procedure, based on the "PRISMA" optimization system, in which the optimum separation is achieved systematically and the structures and properties of the substances to be separated are not known. The procedure consists of three stages. In the first of these the basic conditions the stationary phase, vapor phase, and individual solvents are selected with a TLC procedure (generally in nonsaturated chromatographic chambers). In the second stage, the optimum combination of the selected solvents is determined with the PRISMA model. The third part of the procedure includes the selection of the development mode (circular, linear, or anticircular); the selection of an appropriate forced-flow chromatographic technique (over-pressured layer chromatography or rotation planar chromatography) with high-performance thin-layer chromatographic plates; the transfer of the optimized mobile phase to the various analytical, planar, or column preparative liquid chromatographic techniques; and the selection of the operating conditions. For practical reasons, the optimization process is presented with the help of flow charts.     

Semi-Preparative High-Performance Liquid Chromatographic Separation of Potato Glycoalkaloids

Abstract

A semi-preparative high-performance liquid chromatographic method has been developed to separate crude mixtures of the potato glycoalkaloids α-chaconine, α-solanine, commersonine and demissine. Milligram quantities of each substance can be obtained within an 8 hour period. A Zorbax semi-preparative NH₂ column and a solvent system of tetrahydrofuran-water-acetonitrile (55:20:25) were employed for the separation. The flow rate was 1.0 ml/min. Glycoalkaloid separations were monitored using both refractive index and ultraviolet detection (215 nm). Further analyses of these glycoalkaloids were done using analytical high-performance liquid chromatography (HPLC) and thin-layer chromatography (TLC) to check compound purity and identity.     

OPLC and HPTLC methods in physicochemical studies of a new group of antimycotic compounds

In this study two high-performance methods [overpressured layer chromatography (OPLC) and high-performance thin-layer chromatograpy (HPTLC) under reversed-phase conditions] are used to evaluate the hydrophobicity properties of newly synthesized bioactive compounds. The retention behavior of 24 2-(2,4-dihydroxyphenyl)benzothiazoles are examined for acquisition of log k data. With water-methanol mixtures as the mobile phases, the concentration range in which the correlation between log k and methanol content is linear is established for both methods and used to determine the hydrophobicity parameters of log kw by linear extrapolation. The effect of substituents on retention constants is quantitated by using the group contribution parameters (tauw). The results suggest the use of OPLC methods for quick analysis of physicochemical properties of a large number of organic compounds.     

Development of a New,Rapid, and Sensitive Validated High-Performance Thin-Layer Chromatographic Method for the Estimation of Berberine in Tinospora cordifolia

JPC – Journal of Planar Chromatography – Modern TLC - A straightforward, delicate, and fast elite thin-layer chromatographic system (high-performance thin-layer chromatography [HPTLC])...     

Separation,Identification, and Quantification of N-Acetyl Cilastatin in Human Urine

Abstract

A new high-performance liquid chromatographic method coupled with anion-exchange sample extraction has been developed for the isolation and quantification of N-acetyl cilastatin from human urine samples. N-Acetyl cilastatin, isolated from urine after I.V. administration of cilastatin, was converted to dimethyl esters, and characterized by thin-layer chromatography and mass spectrometry. The detection limit of the assay was 5 μg/ml in urine. The method was shown to be linear, reproducible and reliable for the quantification of N-acetyl cilastatin in urine from four human subjects given I.V. doses of cilastatin alone and together with 250 and 1,000 mg of imipenem.     

Drug detection in hair by chromatographic procedures.

This article reviews the analysis of 31 drugs and drug metabolites in human hair by thin-layer chromatography, high-performance liquid chromatography, gas chromatography, gas chromatography-mass spectrometry and mass spectrometry. The most important detection method after chromatographic separation of the components is the mass spectrometry because of its sensitivity and specificity. Washing steps to exclude external contamination, extraction, derivatization, stationary phases, detection modes and detection limits of the mass spectrometric and gas chromatographic-mass spectrometric procedures are presented in five tables. Additionally, a method for a gas chromatographic-mass spectrometric screening procedure is presented.