Thin-layer Chromatographic Systems Research Articles

Platelet-neutrophil interactions. (12S)-hydroxyeicosatetraen-1,20-dioic acid: a new eicosanoid synthesized by unstimulated neutrophils from (12S)-20-dihydroxyeicosatetraenoic acid.

In the course of a cell-cell interaction, 12-HETE (12-hydroxy-5,8,10,14-eicosatetraenoic acid), the arachidonic acid lipoxygenase product released from stimulated platelets, is metabolized by a cytochrome P-450 enzyme system in unstimulated neutrophils to 12,20-DiHETE (12,20-dihydroxy-5,8,10,14-eicosatetraenoic acid). This report describes time-dependent formation of a new eicosanoid by unstimulated neutrophils exposed to 12-HETE, which is more polar than 12,20-DiHETE (reversed-phase high performance liquid chromatography). Time course studies indicated that the precursor compound of this new eicosanoid was 12,20-DiHETE. This was determined by incubation of purified 12,20-DiHETE with neutrophils, which resulted in a progressive decrease in 12,20-DiHETE as formation of the polar metabolite increased. In the absence of neutrophils, 12,20-DiHETE was quantitatively unchanged. The new metabolite of 12,20-DiHETE was identified as 12-hydroxyeicosatetraen-1,20-dioic acid, based upon its UV spectrum, co-chromatography with a chemically synthesized standard in both high performance liquid chromatography and thin layer chromatography systems, and gas chromatography-mass spectrometry. Formation of 12-HETE-1,20-dioic acid was partially inhibited by 20-hydroxy-LTB4. This indicated that the neutrophil dehydrogenase responsible for further metabolism of 12,20-DiHETE may also be involved in conversion of 20-hydroxy-LTB4 to 20-carboxy-LTB4. The 12,20-DiHETE dehydrogenase enzyme system specifically requires NAD as cofactor and has subcellular components in both cytosolic and microsomal fractions which are synergistic in their activity. These results provide additional evidence for the occurrence of multicellular metabolic events during hemostasis, thrombosis, and the inflammatory response.

Observations on the occurrence and distribution of alkaloids in some genera and species of the tribe Crotalarieae (Fabaceae)

A general survey of alkaloids in the tribe Crotalarieae showed that useful chemotaxonomic data may be obtained. Although the pyrrolizidine alkaloids of Crotalaria L. have been studied in considerable detail, virtually nothing is known for the other, predominantly southern African genera. The genera Dichilus DC., Lebeckia Thunb., Melolobium Eckl. & Zeyh. and Polhillia Stirton were found to contain several alkaloids in sufficient quantities to warrant further investigation. The major alkaloids (presumably all of the quinolizidine type) seem to be characteristic for each of these genera. Aspalathus L., Buchenroedera Eckl. & Zeyh., Lotononis (DC.) Eckl. & Zeyh., Pearsonia Duemmer, Rafnia Thunb. and Wiborgia Thunb. appear to produce much smaller amounts. The possibility of obtaining useful information from these genera seems limited in view of the large quantities of plant material required to enable proper identification of the compounds. Methods of extraction and detection are described. The R f values of the major alkaloids are given for the different thin-layer chromatographic systems used. Some preliminary identifications were confirmed by mass spectrometry.

Identification of tryptophan in leachate of oat hulls (Avena sativa) as a mediator of root growth regulation

Leachate of oat hulls (Avena sativa L. cv. Sang) inhibits root elongation and causes swollen roots and abundant root hair formation. The active substance was isolated by column chromatography and thin layer chromatography (TLC) systems. High performance liquid chromatography (HPLC) revealed 3 peaks, one of which corresponded to the substance responsible. The latter was identified as tryptophan by means of its UV spectrum, amino acid analysis, nuclear magnetic resonance spectrometry (NMR) and mass spectrometry (MS).

ADP-ribosylation of membrane proteins by bacterial toxins in the presence of NAD glycohydrolase

The ADP-ribosylation of membrane G proteins is difficult to achieve in tissues that are rich in membranebound NAD glycohydrolase (NAD + glycohydrolase, EC 3.2.2.5). For many animal species this problem can be surmounted by inhibiting NAD hydrolysis with a combination of the anti-tuberculous drug, isonicotinic acid hydrazide, and the NAD analog, 3-acetylpyridine adenine dinucleotide, which act synergistically. In their presence, the ADP-ribosylation of cholera and pertussis toxin substrates reach plateau levels even with only 10 μM NAD. Although 3-acetylpyridine adenine dinucleotide acts as a weak substrate for the toxins, it is simple to estimate its contribution to the ADP-ribosylation and thus to determine the total amount of ADP-ribosylation substrate present in a tissue sample. NAD glycohydrolases that are insensitive to isonicotinic acid hydrazide are also less sensitive to 3-acetylpyridine adenine dinucleotide, but may be inactivated by dithiothreitol. Isonicotinic acid hydrazide adenine dinucleotide, the product of an exchange reaction eatalysed by NAD glycohydrolase, runs with NAD in most thin-layer chromatographic systems. It can be separated from NAD, and quantitated if the chromatographic solvent contains benzaldehyde. Isonicotinic acid hydrazide itself inhibits NAD glycohydrolase. It need not first be converted into isonicotinic acid hydrazide adenine dinucleotide.

Applicability of forced-flow planar chromatographic methods for the separation of enantiomers on chiralplate®

The separation of enantiomers using forced-flow planar chromatographic techniques such as overpressured layer chromatography and various rotation planar chromatographic methods such as ultra-microchamber rotation planar chromatography and microchamber rotation planar chromatography is reported for the first time. For circular and linear overpressured layer chromatography and ultra-microchamber rotation planar chromatography, the thin-layer chromatographic (TLC) mobile phase system from chromatography in an unsaturated chamber could be applied directly. In overpressured layer chromatography, a prerun had to be made before starting the separation in order to eliminate the disturbing zone. To achieve better migration of the α-front on the chiralplate® in ultra-microchamber rotation planar chromatography an area of 10–20 cm 2 had to be wetted with a component of the mobile phase in which the substances did not migrate. The TLC mobile phase used in a saturated chamber exhibits similar properties when transferred to microchamber rotation planar chromatography. Employing forced-flow planar chromatographic techniques as many as 80 samples can be separated on a single plate. In all instances the separations were better and faster with the forced-flow methods than those achieved with TLC with the same mobile phase.

Analysis of tissue free fatty acids isolated by aminopropyl bonded-phase columns

Gas chromatographic analysis revealed that polyunsaturated fatty acids such as arachidonic acid and total tissue free fatty acids isolated from an aminopropyl bonded-phase column yield a two- to three-fold higher recovery of arachidonic acid as compared to those isolated from thin-layer chromatographic plates. This method was further improved by packing the aminopropyl bonded phase in glass columns, since the glass column significantly eliminated the other contaminants (from polypropylene columns) coeluting with fatty acids in both a neutral lipid thin-layer chromatographic system and on a 5% DEGS-PS column of gas chromatographic analysis. In aminopropyl bonded-phase columns, the standard triglycerides and phospholipids were completely separated from free fatty acids as judged by gas chromatographic analysis. These results warrant the use of an aminopropyl bonded-phase column for the isolation of free fatty acids to obtain better recovery of polyunsaturated fatty acids.

Rapid two-dimensional thin-layer chromatographic system for the separation of multiple steroids of secretory and neuroendocrine interest

Rapid two-dimensional thin-layer chromatographic system for the separation of multiple steroids of secretory and neuroendocrine interest

Thin Layer Chromatographic Analysis of Basic and Quaternary Drugs Extracted as Bis(2-ethylexyl)phosphate Ion-Pairs

Abstract Thin layer chromatography in the normal and reversed phase mode is evaluated as a qualitative analysis method for various basic and quaternary drugs extracted as bis(2-ethylhexyl)phosphate ion-pairs. A combination of two thin layer chromatographic systems, a reversed phase system and a fairly apolar normal phase system, is shown to cover the analysis of drugs with different grades of polarity. Based on that, a method for screening of urine samples for drugs is described.

Detection of mycobacterial lipids in skin biopsies from leprosy patients

Lipids were extracted from small skin biopsies obtained from 5 lepromatous leprosy patients by employing a procedure designed to produce separate non-polar and polar fractions. The defatted tissue was subjected to alkaline hydrolysis and the total fatty acids in the hydrolysate were converted to methyl esters by a phase transfer reaction using iodomethane. The composition of free lipids and methyl esters of fatty acids was analysed by special thin-layer chromatographic systems. The presence of dimycocerosates of phthiocerol A, phthiocerol B and phthiodiolone A, phenolic glycolipid-I, two phosphatidyl inositol mannosides and 2 mycolates − alpha- and keto-mycolate − was demonstrated using reference lipid samples.

A simplified thin-layer chromatography system for the detection of commonly abused basic drugs.

A simple method for the detection of drugs of abuse and their metabolites in urine is described. By extraction of urine (20 mL) with a buffered ToxElut solid phase column all the commonly abused opioid drugs can be detected on a single thin-layer chromatography plate. Location of the spots is achieved by spraying the plate with iodoplatinate following acidification with dilute sulphuric acid. Sensitivity of the order of 1 mg/L is attained for most drugs.

A coupled optical assay for adenine phosphoribosyltransferase and its extension for the spectrophotometric and radioenzymatic determination of 5-phosphoribosyl-1-pyrophosphate in mixtures and in tissue extracts

A reliable assay was developed to characterize crude cell homogenates with regard to their adenine phosphoribosyltransferase activities. The 5-phosphoribosyl-1-pyrophosphate (PRPP)-dependent formation of AMP from adenine is followed spectrophotometrically at 265 nm by coupling it with the following two-stage enzymatic conversion: AMP + H 2O adenosine + P i (5′-nucleotidase); adenosine + H 2O inosine + NH 3 (adenosine deaminase). The same principle was applied to develop a spectrophotometric and a radioenzymatic assay for PRPP. The basis of the spectrophotometric assay is the absorbance change at 265 nm associated with the enzymatic conversion of PRPP into inosine, catalyzed by the sequential action of partially purified adenine phosphoribosyltransferase, commercial 5′-nucleotidase, and commercial adenosine deaminase, in the presence of excess adenine. In the radiochemical assay PRPP is quantitatively converted into [ 14C]inosine via the same combined reaction. Tissue extracts are incubated with excess [ 14C]adenine. The radioactivity of inosine, separated by a thin-layer chromatographic system, is a measure of PRPP present in tissue extracts. The radioenzymatic assay is at least as sensitive as other methods based on the use of adenine phosphoribosyltransferase. However, it overcomes the reversibility of the reaction and the need to use transferase preparations free of any phosphatase and adenosine deaminase activities.

Room-temperature luminescence analysis of drugs with application to the Toxi-Lab drug identification system.

A method of analyzing drugs on the Toxi-Lab thin-layer chromatographic system by solid substrate room-temperature luminescence is described. Three pairs of unresolved drugs: quinine/cimetidine, caffeine/chlordiazepoxide, and phenazopyridine/lidocaine, were studied as model compounds. Their luminescence characteristics were obtained and calibration curves were found to be linear over two orders of magnitude. The effect of the nonluminescent component of each pair on the determination of the luminescent component was found to be negligible. Statistical F-tests showed that the observed differences in the slopes and intercepts of the calibration curves were due to random errors. The method was evaluated by determining quinine in urine samples at the ng level.

Kinin metabolism in human nasal secretions during experimentally induced allergic rhinitis.

We have previously shown that both bradykinin and lysylbradykinin are generated in nasal secretions upon nasal challenge of allergic individuals with appropriate allergen and have suggested that these potent pro-inflammatory peptides may contribute to the pathogenesis of the allergic response. In this study we used a variety of synthetic substrates together with both thin layer and high performance liquid chromatography systems to examine the metabolism of these peptides in nasal secretions obtained by lavage. We now demonstrate that in addition to low levels of angiotensin-converting enzyme, nasal lavages contain an aminopeptidase activity that converts lysylbradykinin to bradykinin, and a carboxypeptidase that removes the C-terminal arginine from bradykinin and lysylbradykinin. The levels of all these activities are significantly increased after allergen challenge of allergic, but not nonallergic, individuals. The aminopeptidase and carboxypeptidase activities present in post-challenge lavages from allergic individuals convert lysylbradykinin to intermediate products (bradykinin and des (Arg10) lysylbradykinin) and eventually to des (Arg9) bradykinin. The nasal carboxypeptidase was activated 475% by 0.1 mM CoCl2 and was inhibited by the carboxypeptidase N inhibitor, MERGETPA (D-L-mercaptomethyl-3-guanidino-ethylthiopropanoic acid) (IC50 = 10 microM). The aminopeptidase activity was not affected by MERGETPA but was potently inhibited by amastatin and bestatin (IC50 = 0.05 microM and 3.0 microM, respectively). The activity of the aminopeptidase against its synthetic substrate was also inhibited by lysylbradykinin (IC50 = 50 microM). Both the carboxypeptidase and aminopeptidase activities had neutral pH optima and were inhibited by o-phenanthroline, but were unaffected by inhibitors of neutral endopeptidases (phosphoramidon) or angiotensin-converting enzyme (Captopril). The Km of bradykinin for the nasal carboxypeptidase was 139 +/- 14 microM (n = 3). We conclude that during the allergic response, nasal secretions contain aminopeptidase and carboxypeptidase activities that convert lysylbradykinin and bradykinin (B2 agonists) to des (Arg9) bradykinin (a B1 agonist). Because the nature of the kinin receptors in the nasal mucosa are currently unknown, it remains to be determined whether this metabolism results in the termination of biologic activity or the production of a biologically active moiety.

Chromatographic methods for the analysis of vancomycin

Four thin-layer chromatographic systems were developed for the separation of vancomycin, related antibiotics and degradation products. Bioautography was suitable for detecting trace amounts of biologically active components. High-performance liquid chromatography was used to examine the composition of vancomycin and other glycopeptide antibiotics and to monitor the stability of vancomycin. Degradation of vancomycin lead to changes in the composition which were not matched by a similar loss of potency.

Variables affecting resolution of lung phospholipids in one-dimensional thin-layer chromatography.

Resolution of the confusion in the literature about the separation of lung phospholipids in thin-layer chromatographic systems has awaited a systematic study of the variables that potentially affect this separation. In this study I show that: incorporation of ammonium sulfate into silica gel "GHL" has a dramatic effect on separation of lung phospholipids; this effect is equally dramatic but different in activated and nonactivated gels; when it picks up moisture, ammonium sulfate-activated gel very rapidly loses its ability to resolve lecithin from phosphatidylinositol; in gel containing ammonium sulfate, small amounts of phosphatidylethanolamine are hydrolyzed to lyso-phosphatidylethanolamine.

High pressure liquid chromatographic determination of dyclonine hydrochloride and its degradation products in cough lozenges and liquids

The local anaesthetic properties of dyclonine hydrochloride, 1-(4-butoxyphenyl)-3-(lpiperidinyl)-1-propanone have been known for some time [l]. Its use in a lozenge formulation has also been described [2]. Yet there is a paucity of analytical procedures for this drug reported in the literature. Thin layer chromatographic (TLC) systems [3,4] and a gas chromatographic (GC) system [5] have been presented but without quantitation. Polarographic characterization procedures [6,7] have also been presented. No decomposition studies or stability results for dyclonine have been reported. Analytical methodology was required to assess the stability of dyclonine hydrochloride and determine its degradation route and products in pharmaceutical preparations used as cough lozenges and syrups. Both GC and high pressure liquid chromatographic (HPLC) approaches were investigated and found feasible. However, the GC approach necessitated pH sensitive extractions as the free base prior to injection. This paper presents HPLC systems and procedures developed for monitoring the stability of dyclonine hydrochloride in formulations and the results of the stability studies. In addition, the structures of some of the probable degradation products have been elucidated and described herein.

Thin-layer chromatography--the forgotten alternative for the quantitative determination of steroids.

We have developed a high performance thin layer chromatography (HPTLC) system for quantitative determination of androgens, corticosteroids, mineralocorticoids and gestagens on silicagel KG-60 HPTLC-plates with different solvent systems. A complete separation of androgens, gestagens and metabolites was achieved with dichlormethane/cyclohexane/acetone (70:25:5). Corticosteroids, mineralocorticoids and their derivatives were completely separated with diethylether/isooctane/isopropanol (70:25:5). The quantitative in situ fluorescence determination was carried out after post-chromatographic derivatization with cinnamic aldehyde, 4-dimethylaminobenzaldehyde and sulfuric acid. The sensitivity of detection was found between 500 pg and 1 ng per spot. The steroid metabolism as catalysed by rat liver microsomal oxidoreductases was measured by these procedures, and was compared with determination of steroids by gas chromatography (GC). According to HPTLC, steroids were reduced by NADPH-5 alpha-reductase (EC 1.3.1.4) in the order progesterone greater than testosterone greater than aldosterone greater than cortisol greater than corticosterone. The enzyme activities as measured by HPTLC agree well with those obtained by GC (r = 0.94). When turnover of enzyme assays, speed of determination, detection limit, application to labile steroids and costs of steroid determination are considered, all points speak in favour of HPTLC.

A new synthetic method for 99mTc labeled N-Pyridoxyl-5-methyltryptophan as a hepatoma imaging agent

N-Pyridoxyl-5-methyltryptophan (5-PMT) was synthetized by a simplified method using sodium borohydride for the reduction of a Schiff base of pyridoxal and 5-methyltryptophan. Lyophilized kits containing 5-PMT, stannous chloride and l-(+)-ascorbic acid were prepared and labeled to afford 99mTc-5-PMT with 96% or higher radiochemical purity analysed by two thin-layer chromatographic solvent systems. 99mTc-5-PMT showed a rapid blood clearance, a faster hepatobiliary transit and a lower renal retention in comparison with 99mTc-5-EHIDA in rats. Eleven (61%) of 18 patients with histologically confirmed hepatocellular carcinoma showed positive images at 2 to 5 h after i.v. injection. The smallest tumor that could be identified was 2 cm in diameter with the best tumor/liver ratio of 4. In conclusion, 99mTc-5-PMT synthesized by sodium borohydride reduction shows great promise as a useful hepatoma imaging agent.

Cytokinin Translocation and Metabolism in Lupin Species. I. Zeatin Riboside Introduced Into the Xylem at the Base of Lupinus angustifolius Stems

The principal cytokinins in xylem exudate of blue lupin (Lupinus angustifolius L.) identified by radioimmunoassay and mass spectrometry were zeatin riboside (ZR), dihydrozeatin riboside and zeatin, but cytokinin O-glucosides and nucleotides were also present. Radioactive [3H]ZR was supplied to the transpiration stream of both derooted and intact blue lupin plants. The distribution of methanol-extracted radioactivity throughout the shoot and the identities of 3H-labelled metabolites were determined; for the latter work, a new bilayer thin-layer chromatography system was developed. In derooted plants supplied with ZR for about 20 h, radioactivity per unit fresh weight of tissue was greatest in the peduncle, stem and axillary shoots and least in the seed. Differences in metabolism were more marked between tissue types than within tissues of different maturity. The principal metabolites in derooted plants (ZR supplied for up to 24 h) were: in laminae, O-glucosyldihydrozeatin, lupinic acid, dihydrozeatin riboside and adenosine; in petioles, dihydrozeatin riboside and adenosine; in pod walls, cytokinin nucleotides, dihydrozeatin riboside, adenine and adenosine; in developing lateral shoots, cytokinin nucleotides; in stem, dihydrozeatin riboside, dihydrozeatin and cytokinin nucleotides. Apart from adenosine, no metabolites could be identified with certainty in seed. In experiments with intact plants conducted over a longer period (plants extracted 2 days after 30 h uptake), metabolism was more extensive but, in all tissues except seed, low percentages of radioactivity were still due to metabolites with an intact zeatin moiety (most frequently O-glucosyldihydrozeatin and lupinic acid). Free zeatin and dihydrozeatin were not found in any tissue and their ribosides were detected with certainty only in pod walls (<2% of extracted 3H). Extraction with 0.5 N KOH after extraction with methanol yielded, on average, an additional equal amount of radioactivity due to nucleotides of adenine and guanine. Features of the translocation studies include: (1) the direct lateral movement of ZR and/or related cytokinins from xylem to bark which was established by bark ringing experiments; (2) the relatively high level of radioactivity per unit tissue fresh weight in developing lateral shoots; (3) the lack of appreciable movement to the seed although the cytokinin reached the pod walls and was partially conserved there; (4) the disproportionately high retention of radioactivity by the petioles which increased with leaf age; (5) changes in ZR metabolites in laminae associated with age; (6) the detection of a new nucleotide metabolite of ZR in pod walls, bark and lateral shoots; this appears to be a transient metabolite with an intact ZR moiety and may be involved in ZR uptake and/or transport. The studies of ZR translocation and metabolism are discussed in relation to sequential leaf senescence, the proposal that developing seeds accumulate xylem cytokinins, and the origin of phloem cytokinins.

Effects of phenylbutazone and oxyphenbutazone on acidic drug detection in high performance thin layer chromatographic systems

Interference or “masking” in thin layer chromatography occurs when the presence of one drug on a thin layer plate physically obscures or interferes with the detection of another drug. We investigated the ability of phenylbutazone and oxyphenbutazone to mask or interfere with the detection of acidic drugs of high performance thin layer chromatography. Of 20 acidic drugs called “positive” since 1981 by laboratories affiliated with the Association of Official Racing Chemists, 16 did not comigrate with phenylbutazone or oxyphenbutazone and could not, therefore, be masked by these agents. Three medications (diclofenac, fenoprofen, ibuprofen) were potentially masked by phenylbutazone and one (sulindac) was potentially masked by oxyphenbutazone. These agents were therefore administered to horses to determine whether or not their metabolites would allow their detection. In each case, metabolites of these agents were detectable for at least 24 hr after drug administration and detection was not interfered with by phenylbutazone or oxyphenbutazone. These results suggest that these 20 acidic drugs should be readily detectable in postrace urines of horses in the presence of phenylbutazone either as the parent drug or by virtue of the easily distinguishable metabolites that each agent possesses. There is, therefore, no reason to believe that the agents tested in this study can be effectively masked or interfered with by phenylbutazone or its metabolites in equine urine.