Separation of brain monosialoganglioside molecular species by high-performance liquid chromatography

A method for the separation of molecular species of brain monosialogangliosides by high-performance liquid chromatography is described. GM4, GM3, GM2, and GM1 were purified from human brain and their individual molecular species were separated on a C18 reversed-phase column. Peaks were identified by mass spectrometry of the intact ganglioside, by gas-liquid chromatography of the fatty acids, and by high-performance liquid chromatography of the long chain bases. A characteristic elution sequence of molecular species permitted their identification based upon their retention times on the reversed-phase column.     

A high-performance liquid chromatography method for the simultaneous assay of diaminopimelate epimerase and decarboxylase

A sensitive and comparatively simple method for the assay of diaminopimelate (DAP) decarboxylase, which simultaneously monitors DAP epimerase activity, in the reverse of the biosynthetic direction, is described. The substrate, meso-DAP and products LL-DAP and L-lysine are derivatized with o-phthaldialdehyde and resolved by reversed-phase high-performance liquid chromatography. Separation is achieved on a Spherisorb C18 column using a gradient elution system. This technique offers a high degree of sensitivity as the detection method described can measure picomole quantities of substrate and products.     

Column-switching high-performance liquid chromatographic detection of pholcodine and its metabolites in urine with fluorescence and electrochemical detection

A sensitive and selective method for the detection of pholcodine and its metabolite morphine in urine using high-performance liquid chromatography is described. It involves on-line clean-up of urine on a trace enrichment column packed with a polymeric strong cation-exchange material. Pholcodine and its metabolites were separated on two analytical columns with different selectivities. Pholcodine was detected by a fluorescence detector and morphine was detected electrochemically. One system, based on reversed-phase chromatography, applied a polystyrene—divinylbenzene column and gradient elution. The other system was based on normal-phase chromatography with a silica column and isocratic elution. Morphine was confirmed to be a metabolite of pholcodine by reversed-phase chromatography and electrochemical detection. Two unidentified metabolites of pholcodine were separated from pholcodine by normal-phase chromatography and detected by fluorescence detection.     

An approach to enhancing coverage of the urinary metabonome using liquid chromatography-ion mobility-mass spectrometry

The potential of drift tube ion mobility (IM) spectrometry in combination with high performance liquid chromatography (LC) and mass spectrometry (MS) for the metabonomic analysis of rat urine is reported. The combined LC-IM-MS approach using quadrupole/time-of-flight mass spectrometry with electrospray ionisation, uses gas-phase analyte characterisation based on both mass-to-charge (m/z) ratio and relative gas-phase mobility (drift time) following LC separation. The technique allowed the acquisition of nested data sets, with mass spectra acquired at regular intervals (65 micros) during each IMS separation (approximately 13 ms) and several IMS spectra acquired during the elution of a single LC peak, without increasing the overall analysis time compared to LC-MS. Preliminary results indicate that spectral quality is improved when using LC-IM-MS, compared to direct injection IM-MS, for which significant ion suppression effects were observed in the electrospray ion source. The use of reversed-phase LC employing fast gradient elution reduced sample preparation to a minimum, whilst maintaining the potential for high throughput analysis. Data mining allowed information on specific analytes to be extracted from the complex metabonomic data set. LC-IM-MS based approaches may have a useful role in metabonomic analyses by introducing an additional discriminatory dimension of ion mobility (drift time).     

Molecular-size determination of xanthan polysaccharide

Hydrodynamic chromatographic separations of xanthan polysaccharide of ultrahigh molecular weight have been obtained by using columns packed with 30-μm, non-porous spheres. From calibration curves of the elution volume versus particle size for spherical, polystyrene latexes, it was found that xanthan is eluted at the same volume as a 0.153-μm diameter sphere. Extremely dilute samples of xanthan (70 p.p.m.) were injected to preclude self-association and aggregate formation. Detection at these low concentrations was accomplished by tagging the xanthan with a fluorescein derivative and using a flow-through fluorometer detector. Flow rates of 1 mL/min yielded run times of ～7 min. Comparison of the accepted molecular conformation of xanthan—a rigid rod-like molecule—with the apparent molecular volume from the spherical-latex calibration indicates that the xanthan molecules are substantially oriented by the flow field in the chromatography column.     

High-performance liquid chromatographic determination of free and total polyamines in human serum as fluorescamine derivatives

A highly sensitive and simple fluorimetric method for the determination of free and total polyamines, spermidine, spermine, putrescine and cadaverine, in human serum by high-performance liquid chromatography is described. The polyamines, obtained after clean-up of deproteinized serum by Cellex P column chromatography, are converted to their fluorescamine derivatives in the presence of nickel ion which inhibits the reaction of interfering amines with fluorescamine, and the derivatives are separated simultaneously by reversed-phase chromatography (LiChrosorb RP-18) with a linear gradient elution. The lower limits of detection are 10 and 5 pmole for spermine and the others in 0.5 ml of serum, respectively.     

Facile analysis and purification of deblocked N-terminal pyroglutamyl peptides with a strong cation-exchange sulfoethyl aspartamide column

A high-performance strong cation-exchange Sulfoethyl Aspartamide column was used to analyze and purify five N-terminal pyroglutamyl peptides after treatment with Pyroglutamate Aminopeptidase. The resulting deblocked N-1 peptides possess an increased positive charge and are therefore retained to a greater extent by the column. Salt gradient elution in a pH 3 mobile phase was then used to recover the desired peptides and the purified deblocked peptides were directly subjected to N-terminal sequence analysis. The same digests were also chromatographed on a C18 reversed-phase column using standard trifluoroacetic acid-acetonitrile gradient elution. The elution order for the parent peptide and the N-1 peptide on the reversed-phase column was reversed from that on the Sulfoethyl Aspartamide column and the resolution of the two peptides obtained on the reversed-phase column was less than that observed on the cation-exchange column. In addition, the Sulfoethyl Aspartamide column was shown to be useful to monitor the extent of N-terminal glutamine cyclization formed during peptide purification and storage.     

Analysis of aminophospholipid molecular species by high performance liquid chromatography

A new method is described for the separation of individual molecular species of the aminophospholipids, phosphatidylethanolamine and phosphatidylserine. Trinitrobenzene-sulfonic acid was used to derivatize both aminophospholipids and the derivatives were purified by thin-layer chromatography. A reversed-phase high performance liquid chromatography technique was developed to separate and quantify individual molecular species based upon ultraviolet detection of the attached chromophore. The retention times of the molecular species on the C18 reversed-phase column were longer with increasing carbon chain length and decreasing degree of unsaturation of fatty acyl chain. The overall procedure allowed a quantitative recovery of the aminophospholipid species. The lower limit of detection was about 10 pmol and a linear response was observed in the range of 0.1-10 nmol of phospholipid. Using this method, we were able to separate and quantify trinitrophenyl-phosphatidylethanolamine molecular species of both subclasses (diacyl and alkenyl) from human red blood cells and rat brains. Separation of species was confirmed by gas-liquid chromatographic analysis of the fatty acid content of each peak and by thermospray liquid chromatography-mass spectrometry. This new method provides a convenient and sensitive technique for studies of aminophospholipid molecular species composition. Furthermore, it appears to be a useful tool for the analysis of asymmetric distribution of these species in biological membranes.     

A high-performance liquid chromatography assay for asparagine synthetase

A highly sensitive method for assaying asparagine synthetase and its glutaminase activity is presented. The amino acids L-asparagine, L-aspartate, L-glutamate, and L-glutamine, are separated by derivatization with o-phthaldialdehyde followed by reversed-phase high-performance liquid chromatography on an Altex ultrasphere-ODS C18 column. The elution is isocratic and the mobile phase used is 50 mM sodium acetate buffer (pH 5.9) with 30% methanol. This assay can easily detect picomoles of asparagine, which may be difficult to do with the other assays that have been described.     

A silica-based reversed-phase column for single-stranded oligodeoxyribonucleotides

A novel, silica-based, reversed-phase column targeted to the separation of single-stranded oligodeoxyribonucleotides has been developed by grafting monochlorooctadecylsilane onto silica which is a base material of reversed-phase columns (TSK 120A and 120T), in which polychlorosilane is used as a grafting reagent. By applying a shallow gradient elution of an aqueous acetonitrile solution containing 0.1 M ammonium acetate, chromatography with this column produced well-resolved peaks for large samples such as those having bases up to 26.     

High-performance liquid chromatographic separation and isolation of quinidine and quinine metabolites in rat urine

A procedure for the separation and isolation of the urinary metabolites of quinidine and quinine by reversed-phase high-performance liquid chromatography is described. Nine metabolites of quinidine and eight metabolites of quinine were detected in the urine of male Sprague-Dawley rats after a single dose of quinidine or quinine (50 mg kg^?1). Following extraction from urine, the metabolites were separated on either an analytical or a semi-preparative reversed-phase column by gradient elution. After isolation and derivatization, the metabolites were analyzed by gas chromatography and gas chromatography—mass spectrometry.     

Analytical exclusion chromatography

This review summarizes the development of exclusion chromatography, also termed gel filtration, molecular-sieve chromatography and gel permeation chromatography, for the quantitative characterization of solutes and solute interactions. As well as affording a means of determining molecular mass and molecular mass distribution, the technique offers a convenient way of characterizing solute self-association and solute-ligand interactions in terms of reaction stoichiometry and equilibrium constant. The availability of molecular-sieve media with different selective porosities ensures that very little restriction is imposed on the size of solute amenable to study. Furthermore, access to a diverse array of assay procedures for monitoring the column eluate endows analytical exclusion chromatography with far greater flexibility than other techniques from the viewpoint of solute concentration range that can be examined. In addition to its widely recognized prowess as a means of solute separation and purification, exclusion chromatography thus also possesses considerable potential for investigating the functional roles of the purified solutes.     

Differentiation between indoleacetic Acid and the citrus auxin by column chromatography

A gradient elution column chromatography technique and a step-wise technique succeeded in differentiating between IAA and the citrus auxin. IAA was eluted ahead of the citrus auxin in both systems. The highest Avena curvature ever obtained from the citrus auxin occurred after the auxin had passed through the 2 purification techniques and a paper chromatography step. This is probably due to the elimination of inhibitors. Fluorometric assay, Ehrlich's reaction, thin-layer chromatography, and biological assay were used for the detection of IAA or citrus auxin in the column eluates.     

Separation of peptides dissolved in a sodium dodecyl sulfate solution by reversed-phase liquid chromatography: removal of sodium dodecyl sulfate from peptides using an ion-exchange precolumn

Separation of peptides by reversed-phase liquid chromatography is significantly affected by sodium dodecyl sulfate (SDS) in the sample solution. The strongly acidic group of SDS binds to the reversed-phase column where it serves as an ion exchanger and retards the elution of peptides. By using a DEAE precolumn connected in series to a reversed-phase column, the interference of SDS in the separation of peptides by reversed-phase chromatography can be significantly diminished. This simple method is applicable to the separation of peptide mixtures obtained by digestion of proteins extracted from SDS-polyacrylamide gels. Peptide production with some proteases in the presence of SDS was examined using the present method. Lysylendopeptidase was suitable for digestion in the presence of SDS, but V8 protease was not.     

Monosaccharide determination of glycoconjugates by reverse-phase high-performance liquid chromatography of their phenylthiocarbamyl derivatives.

A method for the determination of neutral sugars and hexosamines present in glycoconjugates by reverse-phase high-performance liquid chromatography (HPLC) of their phenylthiocarbamyl (PTC) derivatives has been developed. After acid hydrolysis, neutral sugars are converted to glycamines by reaction with ammonium acetate in the presence of sodium cyanoborohydride and are subsequently derivatized with phenylisothiocyanate, while the hexosamines present in the same hydrolysate, after separation on Dowex 50, are treated directly with this reagent. HPLC of the PTC-glycamines of the neutral sugars is performed on Microsorb C18 in an isocratic manner while chromatography of the PTC-hexosamines employs a Pico-Tag column with gradient elution to achieve separation from the PTC-amino acids. The procedure has proven to be highly sensitive, requiring as little as picomole amounts for the chromatographic step; monosaccharide compositions determined on glycoproteins and glycopeptides by this method were found to compare favorably to those previously obtained by other techniques.     

Titanium dioxide as a chemo-affinity solid phase in offline phosphopeptide chromatography prior to HPLC-MS/MS analysis

We have developed a new offline chromatographic approach for the selective enrichment of phosphorylated peptides that is directly compatible with subsequent analysis by online nano electrospray ionization tandem mass spectrometry. In this technique, a titanium dioxide (TiO2)-packed pipette tip is used as a phosphopeptide trap that acts as an offline first-dimension separation step in a two-dimensional chromatography system. This is followed by online nano reversed-phase high-performance liquid chromatography. Here, we present suitable methods for enrichment, optimized separately for each step: sample loading, washing and elution from the TiO2-filled tips. To increase the trapping selectivity of the TiO2 column, we used the sodium salt of 1-octanesulfonic acid combined with 2,5-dihydroxybenzoic acid as ion-pairing agents and displacers for acidic peptides. These agents also improve the binding of phosphorylated peptides and block the binding of non-phosphorylated ones. This enrichment procedure takes 30 min, followed by a 100-min HPLC program, including washing and an elution gradient.     

Quantification of lysophosphatidic acids in rat brain tissue by liquid chromatography–electrospray tandem mass spectrometry

Lysophosphatidic acid (LPA) is a lipid mediator with multiple biological functions. A highly selective and sensitive liquid chromatography–tandem mass spectrometry (LC/MS/MS) method was developed for the determination of LPAs (16:0 LPA, 18:0 LPA, 18:1 LPA, 20:4 LPA) in rat brain cryosections. After partitioning the LPAs from other lipophilic material present in the tissue with a liquid–liquid extraction, a reversed-phase column and ion pair technique was used for separating analytes with a gradient elution. An internal standard (17:0 LPA) was included in the analysis. Detection and quantification of the LPAs were carried out with a triple quadrupole mass spectrometer using negative electrospray ionization (ESI) and multiple reaction monitoring (MRM). The artificial formation of LPAs from lysophosphatidylcholines during the sample preparation procedure and instrumentation was carefully studied during the method development. The method was validated; acceptable selectivity, accuracy, precision, recovery, and stability were obtained for concentrations within the calibration curve range of 0.02–1.0 μM for LPAs. The quantification limit of the assay was 54 fmol injected into column for each LPAs. The method was applied to comparative studies of LPA levels in rat brain cryosections after the various chemical pre-treatments of the sections.     

Measurement of resiniferatoxin in serum samples by high-performance liquid chromatography

A novel and simple method of extraction, separation, identification and quantification of resiniferatoxin (RTX) in serum samples is reported. Human serum and whole blood were treated with acetonitrile to denature proteins, such as orosomucoid, and the soluble fraction was passed through a reversed-phase C18 cartridge. RTX eluted from the cartridge was quantified by high-performance liquid chromatography (HPLC) using a reversed-phase C18 column. Reproducible recovery of RTX and tinyatoxin, an internal standard, from serum was achieved. Isocratic elution with 62% acetonitrile provided a suitable retention time without interfering peaks eluting near the analyte. Therefore, the procedure described provides a useful assay for determination of serum RTX pharmacokinetic parameters.     

High-sensitivity phenylthiohydantoin amino acid analysis using conventional and microbore chromatography

Reversed-phase microbore high-performance liquid chromatography was investigated for high-sensitivity analysis of phenylthiohydantoin (PTH) amino acids. A mixed nitrile alkylsilane bonded phase was developed and ternary gradient elution conditions were devised for resolution 150 × 4.6 mm I.D. column and transferred to a 150 × 1 mmI.D. microbore column. The performance of these columns was evaluated in terms of PTH amino acid resolution, enhanced sample detectability, and retention time precision. For this work a general purpose high-performance liquid chromatograph was modified to reduce extra column band broadening and a preformed gradient elution technique was developed to achieve rapid analysis times at microbore flow-rates. The microbore high-performance liquid chromatographic system is useful for high-sensitivity analysis of PTH amino acids in micro-sequencing applications.     

Partial purification of a specific inhibitor of the insulin-like growth factors by reversed-phase high-performance liquid chromatography

We report here preliminary data using reversed-phase high-performance liquid chromatography for the purification of a specific inhibitor (a molecular weight 16,000–18,000 protein) of the insulin-like growth factor (IGF) or somatomedin family. Crude inhibitor prepared from Cohn fraction IV-1 of human serum was first partially purified using an IGF/CH-Sepharose 4B affinity column. Following elution of the bound inhibitor and resuspension in 0.1% aqueous trifluroacetic acid (mobile phase A), it was injected (100 μl; 2.0 mg protein) onto a Brownlee Aquapore RP-300 column. Application of a linear gradient from 0% to 100% mobile phase B (45% isopropanol−0.1% trifluoroacetic acid) resulted in elution of two peaks of inhibitor activity between 31% and 34% isopropanol associated with a major homogeneous protein peak and a minor heterogeneous protein peak. No inhibitor was recovered when an acetonitrile gradient was used instead of isopropanol, indicating that the inhibitor is very hydrophobic. These data suggest that high-performance liquid chromatography offers a simple procedure for the potential purification of IGF inhibitor(s) from normal human serum.