Isolation of nonactic acids fromStreptomyces griseus fermentation broth by thin-layer and high-performance liquid chromatography

Nonactic acid, homononactic acid and their 2-diastereoisomers were isolated by thin-layer chromatography on silica gel and by high-performance liquid chromatography on a reversed phase from the fermentation broth ofStreptomyces griseus, and identified by¹H and¹³C NMR spectroscopy.     

Isolation of brain monoamines using high-performance reversed-phase chromatography

The content of monoamines, their precursors and metabolites was measured in brain specimens weighing several milligram by ion-pair high performance liquid chromatography on reversed phase microcolumns together with electrochemical detection. The properties of different sorbents are compared and the choice of a mobile phase is discussed. The technique of column packing and preparation of brain samples are described.     

Isolation of alpha-latrotoxin using high-performance liquid chromatography methods

alpha-Latrotoxin, a main toxic component of the Latrodectus mactans tredecimguttatus venom is a large polypeptide with molecular weight of 130 KDa. A rapid method is suggested for isolating this protein using high-effective liquid chromatography on chromatograph FPLC ("Pharmacia", Sweden). The isolated protein does not differ from the previously described alpha-latrotoxin in the main physicochemical parameters as well as in physiological properties.     

Separation and quantitation of peroxidized phospholipids using high-performance thin-layer chromatography with tetramethyl-p-phenylenediamine detection

A simple method for the selective determination of phospholipid hydroperoxide (PLOOH) families in complex lipid populations has been developed. Referred to as HPTLC-TPD, the method is based on PLOOH separation by normal-phase high-performance thin-layer chromatography, followed by spray detection with N,N,N',N'-tetramethyl-p-phenylenediamine and densitometric scanning of the purple bands. Parental phospholipids and alcohol analogues are unreactive. Calibration curves, dynamic ranges, and detection limits were established for hydroperoxide standards prepared from phospatidylcholine, phosphatidylserine, phosphatidylethanolamine, and cardiolipin. For all PLOOH classes, responsiveness was linear out to at least 10 nmol of sample load, the detection limit being 0.1-0.2 nmol. HPTLC-TPD data were validated by subjecting duplicate samples to more complex column chromatography with reductive-mode electrochemical detection. General applicability of the new technique was demonstrated using lipid extracts from two test systems: (i) photoperoxidized liposomal membranes and (ii) tumor cells that had been oxidatively stressed with the respiratory inhibitor antimycin A. HPTLC-TPD provides a convenient, specific, and highly sensitive means for quantifying individual PLOOH families in complex natural mixtures.     

Fluorodensitometric evaluation of gentamicin from plasma and urine by high-performance thin-layer chromatography

High-performance thin-layer chromatographic (HPTLC) analysis of gentamicin by in situ fluorodensitometric evaluation of its 4-chloro-7-nitrobenzo-2-oxa-1,3-diazole (NBD-Cl) derivative is presented. The aminoglycoside components separated on silica gel plates using chloroform–methanol–20% ammonium hydroxide (2.4:2.2:1.5, v/v/v) as the mobile phase were reacted with NB-Cl to yield highly fluorescent derivatives. The calibration curves of gentamicin in water, plasma and urine were linear in the range 40–200 ng. The mean values of intercept, slope and correlation coefficient were 16.82±0.473, 6.83±0.015 and 0.9968±0.0017 for standard curves in water, 17.35±0.375, 6.85±0.018 and 0.9941±0.0012 for standard curves in plasma and 14.35±0.286, 6.86±0.002 and 0.9933+0.0011 for standard curves in urine respectively. The analytical technique was validated for within-day and day-to-day variation. The results indicate that HPTLC, coupled with in situ fluorodensitometry, is a reliable and valuable technique for quantitative analysis of the bulk drug gentamicin and gentamicin from urine and plasma.     

Rapid and sensitive detection of benzodiazepines and zopiclone in serum using high-performance thin-layer chromatography

We developed a rapid and sensitive method of identifying benzodiazepines and zopiclone in human serum using high-performance thin-layer chromatography (HPTLC). These drugs were developed and separated on plates within 8–11 min and detected by means of UV radiation and colour. Each drug was accurately identified by means of the values of R_F × 100 and the spot colour in three systems. The detection limit of the benzodiazepines in serum was 0.1-0.4 μg/ml, except for cloxazolam and haloxazolam. The sensitivity was increased about ten-fold over the conventional method. These results suggested that the HPTLC system is useful for the initial detection and identification of these drugs in emergencies.     

Simple purification of aromatic -amino acid decarboxylase from human pheochromocytoma using high-performance liquid chromatography

We purified aromatic -amino acid decarboxylase (AADC) homogeneously and rapidly from human pheochromocytoma using high-performance liquid chromatography. HPLC with gel permeation and hydrophobic columns was highly effective, and the entire purification could be finished within 3 days. Purified AADC showed a single band with an M_r of 50,000 on sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and decarboxylated -3,4-dihydroxyphenyl-alanine, -5-hydroxytryptophan, and -threo-3,4-dihydroxyphenylserine (a synthetic precursor of natural norepinephrine). Amino acid analysis of purified AADC was performed.     

Simple purification of aromatic L-amino acid decarboxylase from human pheochromocytoma using high-performance liquid chromatography

We purified aromatic L-amino acid decarboxylase (AADC) homogeneously and rapidly from human pheochromocytoma using high-performance liquid chromatography. HPLC with gel permeation and hydrophobic columns was highly effective, and the entire purification could be finished within 3 days. Purified AADC showed a single band with an Mr of 50,000 on sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and decarboxylated L-3,4-dihydroxyphenylalanine, L-5-hydroxytryptophan, and L-threo-3,4-dihydroxyphenylserine (a synthetic precursor of natural norepinephrine). Amino acid analysis of purified AADC was performed.     

Isolation of alpha 1-acid glycoprotein from human plasma using high-performance liquid chromatography

A method for the rapid isolation of purified alpha 1-acid glycoprotein (AGP) from small volumes of human plasma using HPLC has been developed. The method involves preparation of the seromucoid fraction of plasma by sequential perchloric acid and phosphotungstic acid precipitations, followed by chromatography on an HPLC TSKG-3000 column. The yield was high (0.75 mg AGP/ml plasma) and the procedure takes less than 1 day. The method lends itself to easy automation and is particularly suitable for isotopic turnover studies requiring multiple plasma samples.     

Purification of a hydrophobic surfactant peptide using high-performance liquid chromatography

A 4- to 6-kDa hydrophobic peptide (SP4-6) was purified from human pulmonary surfactant. Sep Pak Florisil cartridges removed most of the lipids and the 18-kDa peptide. Analytical wide-pore reversed-phase HPLC column separated a single peptide that contained no detectable lipids (less than 1 nmol/2.5 micrograms protein). N-terminal analysis indicated that this peptide was pure, but the N-terminal amino acid was blocked. The peptide was capable of restoring the in vitro surface properties of synthetic phospholipids, which is characteristic of native lung surfactant.     

Histamine-releasing activity. V. Characterization and purification using high-performance liquid chromatography

The production of large quantities of the lymphokine(s) histamine-releasing activity (HRA) and its partial purification by Sephadex G-75 and ion-exchange chromatography on carboxymethyl (CM) Sepharose 6B have been detailed (M. A. Lett-Brown, D. O. Thueson, D. E. Plank, M. P. Langford, and J. A. Grant, Cell. Immunol. 87, 434-444, 1984). Two peaks of activity (HRA I and II) were recovered. Preparations of HRA have now been analyzed by high-performance liquid chromatography (HPLC). Thoracic duct lymphocytes stimulated with 200 U/ml streptokinase were used as a source of HRA. Gel-filtration HPLC on a TSK 3000 column separated HRA into two peaks of activity (10,000-20,000 and 1300 Da). Reverse-phase high-performance liquid chromatography using a Nucleosil C-8 column showed that HRA II (the activity eluted at a conductivity of 18-20 mmho on the CM-Sepharose column) eluted as a single sharp peak, the main protein contaminant being cytochrome c, the carrier protein added to enhance the yield of HRA. High-performance liquid chromatography was found to be a useful analytical tool and may be suitable for the large-scale purification of HRA.     

Isolation of individual amino acids from various microbiological sources using reversed-phase high-performance liquid chromatography

A new method for the preparative isolation of individual amino acids on a milligram scale based on reversed-phase high-performance liquid chromatography (RP-HPLC) after pre-column derivatization with carbobenzoxychloride (ZCl) has been developed. The chromatographic procedure was tested by the investigation of jack bean urease hydrolysate. The method has been applied to the preparative separation of Z-amino acids (from 10 up to 16) obtained from protein hydrolysates of various sources (green microalgae, blue-green algae, halophilic and methylotrophic microorganisms) and was proved to be reliable by the separation of deuterated amino acids (enrichment 97–99%) from Methylobacillus flagellatum (due to the bioconversion of CD₃OD and D₂O). Independent of the biological source of the protein, the amino acids were isolated with high recovery (from 68% up to 89%) and chromatographic purity (from 96% up to 99%). The method was also applied for the isolation of phenylalanine and leucine excreted by amino-acid overproducing microorganisms.     

Isolation and purification of plastocyanin from spinach stored frozen using hydrophobic interaction and ion-exchange chromatography

A new simple three-day procedure for preparative isolation and purification of plastocyanin from spinach stored in the frozen state is described. This procedure is based on batch adsorption on ion-exchange resin, ammonium sulphate precipitation, and purification on a Phenyl-Sepharose hydrophobic interaction column and a single Q Sepharose High Performance ion-exchange column. Approximately 100 mg of plastocyanin with an absorbance ratio A₂₇₈/A₅₉₇ of 1.10±0.02 in the oxidized state was typically obtained from 12 kg of spinach leaves. The purified spinach plastocyanin is shown to be homogeneous to the resolution of free solution capillary electrophoresis.Abbreviations MES 2(N-morpholino)ethanesulfonic acid - Tris Tris(hydroxymethyl)aminomethane - FSCE free solution capillary electrophoresis     

Isolation and purification of angiotensin II using affinity and high-pressure liquid chromatography

Peptides have been found in a variety of tissues including brain. To purify the peptide angiotensin II, a three-step method for the isolation and purification has been developed using extraction, affinity chromatography, and high-pressure liquid chromatography. Angiotensin II antiserum purified by affinity chromatography was covalently coupled to Affi-gel 10 (Affi-gel 10-AB). The efficiency and usefulness of this column for the purification of angiotensin II from biological sources were tested with 125I- and 3H-labeled (Ile5)-angiotensin II added to rat brains prior to extraction. After extraction, the recoveries for both peptides were 74 and 75%, respectively. Recovery after the purification on Affi-gel 10-AB was 84 and 82%. Thirty-two percent of the radioactivity was not retained and 50% of the radioactivity could be eluted with 0.1 M Na citrate buffer containing 1 M NaCl using a stepwise pH gradient. Characterization by HPLC of the unretained radioactivity from the Affi-gel 10-AB column showed one peak for [125I]angiotensin II, coeluting with the [125I]angiotensin II standard and two minor peaks. Only 30% of unretained [3H]angiotensin II could be identified as intact [3H]angiotensin II on HPLC. Both [125I]angiotensin II and [3H]angiotensin II elutable at pH 5.0 and 4.0 on Affi-gel 10-AB could be demonstrated as highly purified [125I]angiotensin II and [3H]angiotensin II on HPLC with a purity of more than 90%. On HPLC, the recovery was 81% for [125I]angiotensin II and 99% for [3H]angiotensin II. The recovery for the entire three-step procedure was about 60%. The loading capacity of the Affi-gel 10-AB column for (Ile5)-angiotensin II was 550 ng.(ABSTRACT TRUNCATED AT 250 WORDS)     

Quantitative high-performance thin-layer chromatography of dansyl derivatives of biogenic amines

A procedure for the separation, detection, and quantification of picomole levels of dansyl derivatives of the biogenic amines, dopamine, norepinephrine, octopamine, and serotonin, has been developed using high-performance thin-layer chromatography. The detection limit is 1 to 2 pmol. Each of the amine derivatives has been detected in insect brain tissue and a solvent system has been developed for the separation and quantification of octopamine in insect tissue samples.