Separation and quantification of muropeptides with high-performance liquid chromatography

About 80 different muropeptides, the subunits which comprise the polymer murein of Escherichia coli, were resolved by high-performance liquid chromatography. The muropeptides were released from isolated murein by complete digestion with muramidase from Chalaropsis spec. The separation method is based on reversed phase chromatography of the sodium borohydride-reduced compounds using ODS (C18) columns and a linear gradient elution with sodium phosphate buffer and methanol as organic modifier. The effect of temperature, pH, ionic strength, and the steepness of the gradient and of different support materials on the separation of the muropeptides was investigated. The new method represents a major improvement over previous methods with respect to resolution, sensitivity, and speed. Analytical as well as preparative separations can be realized. Quantitative analysis of murein composition is achieved by a linear gradient from 50 mM sodium phosphate, pH 4.31, to 75 mM sodium phosphate, pH 4.95, containing 15% methanol for 135 min on a 250 X 4.6 mm 3-micron Hypersil ODS column at 55 degrees C using a flow rate of 0.5 ml/min. With uv detection at 205 nm about 20 micrograms of murein per analysis is sufficient. The detection limit per compound is about 5 ng. A method for the evaluation of the analytical data allowing a convenient comparison of different muropeptide pattern is described.     

Analysis of amino acid phenylthiohydantoins by high-performance liquid chromatography using gradient elution with ethanol

The use of gradient elution with acetate-buffered ethanol for high-performance liquid chromatographic analysis of phenylthiohydantoins (PTHs) is described. The system separates all commonly encountered PTH derivatives in a total analysis time of 20 min on columns of Spherisorb 5 S ODS (4.6 × 250 mm) packed in the laboratory at moderate expense. Experience with routine operation of the method, particularly with regard to column stability is discussed. Ethanol is considerably cheaper and less toxic than previously described solvent systems.     

Purification of fatty acid ethyl esters by solid-phase extraction and high-performance liquid chromatography

We have developed a two-step method to purify fatty acid ethyl esters (FAEE) using solid-phase extraction (SPE), with a recovery of 70±3% (mean±S.E.M.) as assessed using ethyl oleate as a recovery marker from a standard lipid mixture in hexane. The first step of the SPE procedure involves application of a lipid mixture to an aminopropyl-silica column with simultaneous elution of FAEE and cholesteryl esters from the column with hexane. Gas chromatographic analysis of FAEE without interference from cholesteryl esters may be performed using the eluate from the aminopropyl-silica column, thus eliminating the need for an octadecylsily (ODS) column in this case. The FAEE can then be separated from the cholesteryl esters, if necessary, by chromatography on an ODS column and elution with isopropanol-water (5:1, v/v). Both the aminopropyl-silica and ODS columns were found to be effective for up to four uses. To permit isolation of specific FAEE species following isolation of total FAEE by the two-step SPE method, we have also developed a purification scheme for individaal FAEE by high-performance liquid chromatography (HPLC). Thus, this simple method allows for reproducible isolation of total FAEE by SPE and isolation of individual FAEE species by HPLC.     

Determination of scutellarin in rat plasma by high-performance liquid chromatography with ultraviolet detection

A validated high-performance liquid chromatography method is described for the determination of scutellarin in rat plasma using a liquid-liquid extraction and ultraviolet (UV) absorbance detection. The separation used a Diamonsil ODS column (250 mm x 4.6mm i.d., 5 microm particle size) with an isocratic mobile phase consisting of methanol-acetonitrile-50mM dihydrogen ammonium phosphate buffer (22:15:63 (v/v/v), adjusted to pH 2.5 with 1M phosphoric acid). The ultraviolet detector operated at 335 nm. Plasma samples were extracted with ethyl acetate after acidification. The extraction recovery of scutellarin ranged from 68.1 to 80.5%. High selectivity and a low quantitation limit (0.050 microg/ml) were achieved. The linear range was 0.050-12.5 microg/ml, correlation coefficient r=0.9981. The method has a good reproducibility, R.S.D. values were below 7.9% for within-day and between-day precision. The method is simple, rapid, and applicable to preliminary pharmacokinetic studies of scutellarin in rats.     

Automated and simultaneous two-dimensional micro-high-performance liquid chromatographic determination of proline and hydroxyproline enantiomers in mammals

A fully automated 2D-HPLC system employing a microbore-ODS column and a narrowbore-enantioselective column has been developed for the simultaneous enantiomer determination of proline, trans-4-hydroxyproline and cis-4-hydroxyproline in mammals. As a first dimension, a monolithic ODS column of 0.53 mm i.d. showed 3-6 times larger theoretical plate numbers than those of particle-packed ODS columns, and the best enantioseparations were obtained by a Chiralpak QN-2-AX column of 1.5 mm i.d. in the second dimension (separation factors: 1.44-1.83). The R.S.D. values for within-day and dayto-day precisions were less than 5.8%, and the lower limits of quantitation for the D-enantiomers were 1 fmol. The present method was successfully applied to the determination of proline and hydroxyproline enantiomers in the serum and collagen-rich skin tissue. Small amounts of D-proline were found both in the serum (1.57 +/- 0.19 nmol/mL) and in the skin (0.093 +/- 0.015 nmol/mg protein), while the amounts of D-hydroxyproline were smaller than the lower limit of quantitation.     

Simultaneous determination of olanzapine,clozapine and demethylated metabolites in serum by on-line column-switching high-performance liquid chromatography

An automated method for simultaneous routine quantification of the antipsychotic drugs clozapine, olanzapine and their demethylated metabolites is described. The method included adsorption on a cyanopropyl (CPS) coated clean-up column (10 μm; 10×2.0 mm I.D.), washing off interfering serum constituents to waste, and separation on C18 ODS Hypersil reversed phase material (5 μm; 250×4.6 mm I.D.) using acetonitrile–water–tetramethylethylenediamine (37:62.6:0.4, v/v/v) adjusted to pH 6.5 with concentrated acetic acid. UV-detection was performed at 254 nm. The limit of quantification was 10–20 ng/ml. Relative day to day standard variations ranged between 4.5 and 13.5%. The method is suitable for routine monitoring of olanzapine and clozapine including their demethylated metabolites.     

Evaluation of comprehensive multidimensional separations using reversed-phase, reversed-phase liquid chromatography/mass spectrometry for shotgun proteomics

Two-dimensional liquid-chromatographic (LC) separation followed by mass spectrometric (MS) analysis was examined for the identification of peptides in complex mixtures as an alternative to widely used two-dimensional gel electrophoresis followed by MS analysis for use in proteomics. The present method involves the off-line coupling of a narrow-bore, polymer-based, reversed-phase column using an acetonitrile gradient in an alkaline mobile phase in the first dimension with octadecylsilanized silica (ODS)-based nano-LC/MS in the second dimension. After the first separation, successive fractions were acidified and dried off-line, then loaded on the second dimension column. Both columns separate peptides according to hydrophobicity under different pH conditions, but more peptides were identified than with the conventional technique for shotgun proteomics, that is, the combination of a strong cation exchange column with an ODS column, and the system was robust because no salts were included in the mobile phases. The suitability of the method for proteomics measurements was evaluated.     

Separation of tocopherol and tocotrienol isomers using normal- and reverse-phase liquid chromatography

This optimization study for tocopherols and tocotrienols involved both normal- and reverse-phase liquid chromatography using various columns and mobile phases. Normal-phase systems showed elution of the homologs in order of increasing polarity with separation based on methyl substituents on the chromanol moiety. Reverse-phase systems showed class separation based on the saturation of the phytyl side chain; the more saturated tocopherols were retained on the column longer. When the Zorbax ODS was used with an isocratic ternary acetonitrile:methanol:methylene chloride (60:35:5) mixture, the optimized resolution was greater than 2.0 and separation was achieved in less than 13 min, but there was no separation of beta- and gamma-tocopherols. The normal-phase silica and amino columns provided separation of all available isomers with resolution greater than 1.1 and separation times of less than 5.5 and less than 10 min, respectively. Optimized isocratic binary solvent mixtures of hexane:2-propanol were used for silica (99:1) and amino (98:2) columns. Derivative spectra showed differences depending on substituents in the chromanol moiety but not the phytyl side chain. Second- and fourth-derivative spectra gave the best differentiation of the vitamin E isomers.     

Determination of urinary nucleosides by direct injection and coupled-column high-performance liquid chromatography

A coupled-column liquid chromatographic method for the direct analysis of 14 urinary nucleosides is described. Efficient on-line clean-up and concentration of 14 nucleosides from urine samples were obtained by using a boronic acid-substituted silica column (40 mm x 4.0 mm I.D.) as the first column (Col-1) and a Hypersil ODS2 column (250 mm x 4.6 mm I.D.) as the second column (Col-2). The mobile phases applied consisted of 0.25 mol/L ammonium acetate (pH 8.5) on Col-1, and of 25 mmol/L potassium dihydrogen phosphate (pH 4.5) on Col-2, respectively. Determination of urinary nucleosides was performed on Col-2 column by using a linear gradient elution comprising 25 mmol/L potassium dihydrogen phosphate (pH 4.5) and methanol-water (60:40, v/v) with UV detection at 260 nm. Urinary nucleosides analysis can be carried out by this procedure in 50 min requiring only pH adjustment and the protein precipitation by centrifugation of urine samples. Calibration plots of 14 standard nucleosides showed excellent linearity (r > 0.995) and the limits of detection were at micromolar levels. Both of intra- and inter-day precisions of the method were better than 6.6% for direct determination of 14 nucleosides. The validated method was applied to quantify 14 nucleosides in 20 normal urines to establish reference ranges.     

Analysis of oligosaccharides from heparin by reversed-phase ion-pairing high-performance liquid chromatography

An ion-pairing high-pressure liquid chromatography procedure was developed for analysis of mixtures of oligosaccharides generated by nitrous acid cleavage of heparin. Oligosaccharides were eluted from a Hi-Chrom 5S ODS (C18) column using mixtures of acetonitrile and buffers containing 40 mM ammonium phosphate and 1 mM tetrabutylammonium phosphate. Isocratic conditions were developed for optimal separation of a number of individual disaccharides and tetrasaccharides that were characterized previously (M.J. Bienkowski and H.E. Conrad (1985) J. Biol. Chem. 260, 356-365). These isocratic conditions were then coupled to obtain gradient elution conditions for the ion-pairing separations of mixtures of disaccharides and mixtures of tetrasaccharides. A comparison of the elution profiles obtained in the ion-pairing chromatography procedure with profiles obtained by anion-exchange high-pressure liquid chromatography profiles showed markedly better overall resolution by the ion-pairing procedure. As a result of this improved resolution, the new procedure showed the presence of previously unidentified products in the heparin oligosaccharide mixtures.     

Enantiomeric separation of imidazolinone herbicides using chiral high-performance liquid chromatography

Chiral high-performance liquid chromatography (HPLC) is one of the most powerful tools to prepare enantiopure standards of chiral compounds. In this study, the enantiomeric separation of imidazolinone herbicides, i.e., imazethapyr, imazapyr, and imazaquin, was investigated using chiral HPLC. The enantioselectivity of Chiralpak AS, Chiralpak AD, Chiralcel OD, and Chiralcel OJ columns for the three analytes was compared under similar chromatographic conditions. Chiralcel OJ column showed the best chiral resolving capacity among the test columns. The resolved enantiomers were distinguished by their signs of circular dichroism detected at 275 nm and their structures confirmed with LC-mass spectrometric analysis. Factors affecting the chiral separation of imidazolinones on Chiralcel OJ column were characterized. Ethanol acted as a better polar modifier than the other alcohols including 2-propanol, 1-butanol, and 1-pentanol. Although the acidic modifier in the mobile phase did not influence chiral recognition, it was necessary for reducing the retention time of enantiomers and suppressing their peak tailing. Thermodynamic evaluation suggests that enantiomeric separation of imidazolinones on Chiralcel OJ column is an enthalpy-driven process from 10 to 40 degrees C. This study also shows that small amounts of pure enantiomers of imidazolinones may be obtained by using the analytical chiral HPLC approach.     

Determination of lauroyl-indapamide in rat whole blood by high-performance liquid chromatography

A method based on a liquid-liquid extraction procedure followed by high-performance liquid chromatography (HPLC) coupled with UV-visible detection is described and validated for the determination of lauroyl-indapamide in rat whole blood. The blood sample was extracted with diethyl ether after the addition of 10% trifluoroacetic acid (aq.). The chromatographic separation was performed on a Chromasil ODS column, using methanol-acetonitrile-tetrahydrofuran-0.2% trifluoroacetic acid (170:20:15:38, v/v/v/v) as the mobile phase. The UV detection wavelength was set at 240 nm. The extraction recovery of lauroyl-indapamide was ranged from 76.5 to 82.6%, and the calibration curve had a good linearity in the range of 0.048-200 microg/ml (r = 0.9976). The method presents appropriate intra-day and inter-days repeatabilities, showing values below 7.4% in terms of the percentage of relative standard deviation (R.S.D.). The method proposed is simple, rapid and sensitive, being useful for pharmacokinetic studies in rats.     

Automated high-performance liquid chromatographic method for determination of furosemide in dog plasma

An automated high-performance liquid chromatographic method for the determination of the diuretic drug furosemide has been established. Dog plasma was injected directly into a two-column system with a BSA—ODS (ODS column coated with bovine serum albumin) precolumn and a C₁₈ analytical column for the separation of furosemide. The two columns were automatically switched. Furosemide remained trapped on the precolumn while proteins were eluted to waste. After column switching, furosemide was washed onto the analytical column and analysed without interference. The greatest advantage of the method is its easy performance without manual sample preparation; it requires no extraction or deproteinization. The method allows determination of 0.1–10 μg/ml of furosemide with accuracy and precision comparable with previously reported values. The coefficients of variation obtained from replicate measurements of 1 μg/ml and 5 μg/ml samples were 1.65% and 2.40%, respectively. This method was used to measure the plasma levels of furosemide in beagle dogs to whom the drugs was administered, as a reference, in a toxicological study.     

Determination of methotrexate in serum by high-performance liquid chromatography

A sensitive high-performance liquid chromatographic method with ultraviolet detection was developed to quantitate methotrexate in serum-based calibrators, controls and patient samples. Sample clean-up was achieved with C₁₈ Sep-Pak Classic cartridges. The chromatographic separation was accomplished on a 5-μm Ultrasphere ODS Beckman column. 8-Chlorotheophylline was used as an internal standard. The method was validated by recovery, linearity, accuracy and precision studies. Two standard curves were constructed to cover the high and the low ends of the calibrator range (0.05–1.0 μmol/l). Response was found linear over the whole range of the calibrator set with a correlation coefficient of 0.999 and 1.00 for the low-level and the high-level curves, respectively. Accuracy varied from 12% at the lowest level to 1.2% at the highest level. The precision study showed a C.V. of 14.4% at the lowest level and 3.3% at the highest level.     

Variables in the high-performance anion-exchange chromatography of proteins

The effect of mobile phase velocity, separation time, support pore diameter, column length, and temperature on resolution and loading capacity of a new commercially available high-performance anion-exchange support, SynChropak AX-300, has been examined. This material is a macroporous spherical silica of 10 μm particle size with a bonded polymeric amine layer. It was found that the heterogeneity of ovalbumin samples, combined with bovine serum albumin, make them useful probes in evaluation of anion-exchange supports. In the columns of 4.1 mm i.d., the highest resolutions of proteins were achieved at a flow rate of 0.25 ml/min. Up to 10 mg of protein per injection could be applied on a 4.1 × 250 mm AX-300 column with good resolution. Columns of 50 mm length had one-tenth the protein load capacity of a 250-mm column, retaining approximately 75% of the resolution.     

Single polyprenol and dolichol isolation by semipreparative high-performance liquid chromatography technique

A new method of separation of single polyprenols (or dolichols) from a mixture of isoprenoid alcohols is described. Application of a high-performance liquid chromatography (HPLC) apparatus equipped with a semipreparative ODS column resulted in preparation of long-chain (dihydro)polyprenols of high purity (>95%).This approach substantially decreases the time scale of the conventional chromatographical preparative procedure. The method can be widely used in chemical and biochemical projects, where single polyprenols or dolichols are required.     

Preparative ion-exchange high-performance liquid chromatography of bacterial ribosomal proteins

We have developed analytical and preparative ion-exchange HPLC methods for the separation of bacterial ribosomal proteins. Proteins separated by the TSK SP-5-PW column were identified with reverse-phase HPLC and gel electrophoresis. The 21 proteins of the small ribosomal subunit were resolved into 18 peaks, and the 32 large ribosomal subunit proteins produced 25 distinct peaks. All peaks containing more than one protein were resolved using reverse-phase HPLC. Peak volumes were typically a few milliliters. Separation times were 90 min for analytical and 5 h for preparative columns. Preparative-scale sample loads ranged from 100 to 400 mg. Overall recovery efficiency for 30S and 50S subunit proteins was approximately 100%. 30S ribosomal subunit proteins purified by this method were shown to be fully capable of participating in vitro reassembly to form intact, active ribosomal subunits.     

Analysis of pyridine dinucleotides in cultured rat hepatocytes by high-performance liquid chromatography

An isocratic reverse-phase high-performance liquid chromatography method for the separation and quantitation of total pyridine dinucleotides in hepatocyte cultures is described. Cells are extracted with cold 3 M perchloric acid or 0.5 N sodium hydroxide containing 50% (v/v) ethanol and 35% cesium chloride for the determination of the oxidized or reduced pyridine dinucleotides, respectively. Pyridine dinucleotides in the neutralized extracts were separated on an Excellopak ODS C18 (4.6 X 150 mm) column with 0.1 M potassium phosphate, pH 6.0, containing 3.75% methanol as the mobile phase. NAD+ and NADP+ were detected spectrophotometrically at 254 nm. The response was linear from 5 to 4000 pmol with recoveries of NAD+ and NADP+ of 98 and 101.1%, respectively. NADH and NADPH were monitored fluorometrically by activation at 370 nm and emission in the 400-700 nm range. The reduced pyridine dinucleotides had a linear response from 7.5 to 60 pmol with recoveries of NADH and NADPH of 99.4 and 101.3%, respectively. The coefficients of variation for all of the pyridine dinucleotide standards were less than 3.5%.     

Simultaneous determination of benzophenones and gentisein in Hypericum annulatum Moris by high-performance liquid chromatography

The content of the benzophenones, hypericophenonoside, neoannulatophenonoside, annulatophenonoside, annulatophenone, acetylannulatophenonoside and the xanthone derivative gentisein have been determined in aerial parts, leaves, flowers and stems of Hypericum annulatum Moris. Extraction of samples with methanol by magnetic stirring at room temperature allowed a good recovery of analytes (from 90.70% for gentisein to 103.81% for annulatophenonoside) and the precision of the entire procedure was < 6.05%. The subsequent HPLC separation and quantification was achieved using a Hypersil ODS C18 column and UV detection at 290 nm. The mobile phase comprised methanol and 20 mm potassium dihydrogen phosphate (adjusted to a pH of 3.19 with o-phosphoric acid), and gradient elution mode was applied. The detection limits were 0.03, 0.02 and 0.001 microg/mL for hypericophenonoside, acetylannulatophenonoside and gentisein, respectively. The total amounts of the phenolic compounds assayed ranged from 10.92 mg/g in stems to 82.86 mg/g in leaves. Hypericophenonoside was the dominant benzophenone present in the majority of the plant samples, being present in amounts between 7.54 +/- 0.25 mg/g in stems and 64.22 +/- 2.44 mg/g in leaves. Hypericophenonoside accounted for up to 77.50% of the components found in the leaves, whereas annulatophenonoside (6.29 +/- 0.15 mg/g) and acetylannulatophenonoside (8.95 +/- 0.09 mg/g) were detected in much lower quantities. In contrast to leaves, flowers showed a tendency towards higher contents of gentisein (9.35 +/- 0.07 mg/g) and neoannulatophenonoside (4.72 +/- 0.04 mg/g) than the other parts assayed.