An Estimation Technique of Molecular Weights of Bio-Oligomers by Reversed-Phase Liquid Chromatography

Abstract

A new simple and accurate method for molecular weight estimation of oligomers by reversed-phase liquid chromatography was developed and has been proposed to use in the investigation of bio-oligomers. Capacity factors (k′) of 24 peptides and proteins ranging in molecular weight from 200 to 70000 were measured independently under isocratic eluting conditions with slightly different acetonitrile contents in the mobile phase. Profiles of plots of the log k' values of the peptides and proteins against the acetonitrile contents were on straight lines with different slopes. These slopes were characteristic for each oligomer and found to be proportional to the two/thirds power of the molecular weight. This elution behavior of oligomers in reversed-phase liquid chromatography can be explained in terms of the solvophobic theory. The estimated molecular weights of several bio-oligomers were more accurate than those obtained by size-exclusion chromatography. when the range of molecular weights was limited from 10³ to 2×10⁴.     

The Analysis of Insulin-Related Peptides by Reversed-Phase High-Performance Liquid Chromatography

Abstract

This paper describes the use of high performance liquid chromatography (HPLC) for the rapid analysis and purification of insulin-related peptides prepared by solid-phase synthetic procedures. Examples include the bovine insulin C-peptide (34–45), the porcine insulin C-peptide (41–53) and the insulin B-chain fragment (22–27). Chromatographic elution systems containing reducing reagents like B-mercaptoethanol allow the direct analysis of insulin reduction products. Similar systems should allow the rapid analysis of disulphide bond pairing patterns in appropriate polypeptides and proteins either directly or following proteolytic digestion.

Reversed-phase high performance liquid chromatography (RP-HPLC) is a versatile and rapid technique useful for the analysis and purification of biological substances. In a series of recent publication^1–4 we have described methods for the analysis of underivatised amino acids, peptides and proteins on reversed-phase packings using ion-pairing or stationary phase modifying reagents as components of the mobile phase. These studies demonstrated that excellent resolution of closely related peptides can be achieved under a variety of elution conditions. The addition of low levels of phosphoric acid, inorganic or organic phosphates to a mobile phase (generally water-organic solvent mixtures), in particular, allows rapid and reproducible analysis of peptidic compounds with high sensitivity detection at wavelengths down to 190nm^5,6. It is the purpose of this report to show that these chromatographic conditions allow the facile analysis, and purification, of a variety of insulin-related peptides.     

The Use Of Soft Acid Metal Salts Of Carboxylic Acids For Retention Of Hydrophilic Peptides In Reversed-Phase High Performance Liquid Chromatography

Abstract

The effect of various soft acid cation acetate salts on the retention of the peptides Phe-(Gly)_n, where n=1–4, and on phenylalanine, indicates a correlation between the softness character of the cation of the acetate salt used in the mobile phase and retention in reversed-phase high performance liquid chromatography. Inorganic soft acid cation salts did not exhibit the same increased retention properties. By using silver(I) valerate or silver(I) octanoate as ion-pairing reagents in the mobile phase for RP-HPLC, static retention of peptides was achieved.     

Simultaneous Determination of Valsartan and Hydrochlorothiazide in Tablets by High-Performance Liquid Chromatography

ABSTRACT

A method for the simultaneous determination of valsartan and hydrochlorothiazide in tablets is described. The procedure, based on the use of reversed-phase high-performance liquid chromatography, is linear in the concentration range 5.0-10.0 μg ml^?1 for valsartan and 0.5-2.0 μg ml^?1 for hydrochlorothiazide, is simple and rapid and allows accurate and precise results. The limit of detection was 1.0 μg ml^?1 for valsartan and 0.05 μg ml^?1 for hydrochlorothiazide.     

Simultaneous Determination of Andrographolide and Dehydroandrographolide in Chicken Plasma for Application to Pharmacokinetic Studies

A simple, suitable reverse phase liquid chromatographic method was developed for simultaneous determination of andrographolide (1) and dehydroandrographolide (2) in chicken plasma after orally administrating the ultra-fine powder of Andrographis paniculata. Plasma samples were extracted with ethyl acetate. Analysis of the extract was performed on a reversed-phase C18 column with gradient eluent composed of acetonitrile and 0.5% acetic acid. The flow rate was kept at 1 mL min⁻¹ and the detection wavelength was set at 225 and 255 nm for 1 and 2, respectively. All calibration curves showed good linear regression (R ≥ 0.9991). The good precision and recoveries with intra-day and inter-day were 3.2–8.7% and 91.1–98.4%, respectively. The limit of detection was 0.016 µg mL⁻¹ and the limit of quantitation was 0.040 µg mL⁻¹ for the target analytes. This validated method has been successfully applied in the pharmacokinetics study of 1 and 2 after orally administrating the Andrographis paniculata ultra-fine powder to chicken.

     

LC Method for Analysis of Three Flavonols in Rat Plasma and Urine after Oral Administration of Polygonum aviculare Extract

A high-performance liquid chromatographic method has been developed for the simultaneous analysis of the flavonols myricitrin (1), avicularin (2), and juglanin (3) in rat plasma and urine after oral administration of the total flavonoids from Polygonum aviculare. Samples were prepared by solid-phase extraction then separated on a C₁₈ reversed-phase column by use of a mobile-phase gradient prepared from methanol and aqueous formic acid solution. The flow rate was 1 mL min⁻¹. Detection was performed at 254 nm. The calibration range was 11–1,100 μg mL⁻¹ for both 2 and 3 in plasma; in urine the calibration ranges for 1, 2, and 3 were 32–1,600, 11–1,100, and 22–1,100 μg mL⁻¹, respectively. Intra-day and inter-day RSD were less than 4.33 and 3.62% for 2 and 3, respectively, in plasma, and no more than 4.03 and 2.22% for all the analytes in urine. The analytical sensitivity and selectivity of the assay enabled successful application to pharmacokinetic studies of flavonols 1–3 in rats.

     

Prepatellamide A, a new cyclic peptide from the ascidian Lissoclinum patella

A new cyclic peptide, prepatellamide A (1), along with three known cyclic peptides (2)— (4), was isolated from the ascidianLissoclinum patella. The structure of prepatellamide A was determined from one- and two-dimensional¹H and¹³C NMR spectra. The known cyclic peptides were identified as patellamides A (2), B (3) and C (4).     

Determination of Steroids in Human Urine by Micellar Liquid Chromatography

Abstract

A simple and sensitive method for the determination of steroids using micellar liquid chromatography is described. The steroids, including hydroxycorticosterone. corticosterone, northisterone, testosterone, mexdroprogesterone acetate and progesterone, were separated by reversed-phase using a micelles mobile phase following UV detection at 245 nm. The parameters affecting retention of the test solutes such as the concentration of sodium dodecyl sulfate (SDS) and n-butanol-1 in the mobile phase were investigated. It was found that the retention of the solutes was dependent on the composition of mobile phase. The linear calibration plots range from 0.1 to 10 μg ml^?1 in mobile phase containing 5.0 × 10^?2 mol l^?1 SDS/9 % n-butanol-1 at pH 6.0, and the detection limit in order of 0.1 μg ml^?1 was obtained. The proposed method was used for the determination of steroids in urine using direct injection of samples without previous treatment.     

Determination of nitrite and nitrate in Venice lagoon water by ion interaction reversed-phase liquid chromatography

Ion interaction reversed-phase liquid chromatography with octylammonium orthophosphate as the interacting reagent and a reversed-phase C₁₈ column was applied to the identification and determination of nitrite and nitrate in Venice lagoon water. Interference by the high chloride concentration was systematically studied and the results obtained with different column packings were compared. With spectrophotometric detection at 230 nm, nitrite at 0.005 mg 1^?1 can be detected and determined even in the presence of 0.70 M chloride. The dependence of the retention time of nitrite on the chloride concentration was studied for two reversed-phase columns with different packings. Concentrations of 0.30 ± 0.05 mg 1^?1 of nitrite and 0.20 ± 0.05 mg 1^?1 of nitrate were found in Venice lagoon water.     

Preparation of 2-125I-Testosterone and Its Use as Ligand for Radioimmunoassay in Serum

Abstract

A radioiodination technique is described which consists of heating Testosterone (T) with Na¹²⁵I and hydrogen peroxide in acetic acid in sealed vial. The binding capacity of labelled substances was measured after thin layer chromatography of the reaction mixture. One compound was selected for radioimmunoassay on the basis of its binding capacity for anti-T serum. Standard curves utilizing tritiated and radioiodinated T, under identical conditions, were superimposible. Serum hormone concentrations determined by the two tracers were comparable. Accuracy, precision and sensitivity were satisfactory. Mass spectroscopy and nuclear magnetic resonance of the unlabelled iodinated T indicated that iodine is in C-2.     

Rapid isocratic HPLC investigation of radiochemical purity for <Superscript>90</Superscript>Y-DOTATATE

DOTA-conjugated peptides, such as [DOTA⁰, Tyr³]ocreotide (DOTATOC) and [DOTA⁰, Tyr³]octreotate (DOTATATE) can be labeled with radionuclides such as ⁹⁰Y, ¹⁷⁷Lu and ¹¹¹In at high specific activities. These radiolabelled somatostatin analogues are used for peptide receptor radionuclide therapy (PRRT). Currently, radio-high performance liquid chromatography (radio-HPLC) and radio-thin layer chromatography (radio-TLC) are the methods of choice for the analysis of the labeled compounds. In literature, radiochemical purity of the radiolabelled DOTATATE was investigated using gradient reversed-phase radio-HPLC. However, these studies indicate long retention time of the radiolabelled compound of 14.52 min. In our study, a new simple and rapid reversed-phase isocratic system enables the radiochemical purity of the radiolabelled DOTATATE within a few minutes.     

Studies on Iodinated Compounds. IV. High Performance Liquid Chromatographic Determination of Iodide with a Complex of Primary Ammonium Ion - Crown Ether

Abstract

Determination of iodide (I^?) was performed using a complex of primary ammonium ion - crown ether as the counter ion on reversed-phase high performance liquid chromatography (HPLC)_° Retention of the I^? in the present system increased more than the corresponding counter ion system, using the only primary ammonium ion_° The rate of increase of the retention was dependent on pHs', concentration of crown ether and organic solvents of the eluent. Determination of I^? was investigated with hexylamine and 18-crown-6 with a UV detector at 225 nm_° The calibration curve was linear from 25 pmoles to 25 nmoles, and the least detection of I^? was 1 pmole.     

High Performance Liquid Chromatographic Method for Prostaglandin E2 Determination in Human Gastric Juice Without Derivatization

Abstract

A rapid and practical method for the separation and quantitation of PGE² in human gastric juice by high performance liquid chromatography is described. Separation on a reversed-phase column and UV detection allows quantities as little as 20 nanograms of PGE² to be detected. Specificity, sensitivity, high yield and reproducibility make this method particularly suitable for prostaglandin determination in human gastric juice.     

Trace-Level Determination of Phenol by Liquid Chromatography with On-Line Precolumn Technology and Fluorescence Detection

Abstract

A strongly basic anion-exchange resin is used for the trace enrichment and automated sample handling of phenol, with subsequent determination by reversed-phase liquid chromatography with fluorescence detection. Because of the presence of high concentrations of ionic compounds in the water samples tested, phenol is first trapped on a relatively long precolumn filled with a highly hydrophobic packing material; during this step, (in) organic anions which are not retained, are flushed to waste. In the next step, phenol is desorbed from this column at high pH and sorbed in a small zone (“peak compression”) on a short precolumn containing the anion exchanger.

In the analysis of tap and river water samples, the detection limit was found to be 10ppt (1:10¹¹).     

Radioreceptor Assay of Opioid Peptides in Selected Canine Brain Regions

Abstract

A radioreceptor assay using the opioid delta receptor-preferring ligand D- ²ala, D- ⁵leu leucine enkephalin (³H-DADL) and the broader-specificity ligand ³H-etorphine was used to measure five HPLC-purified neuropeptide fractions derived from the peptide-rich fraction of tissue homogenates of nine anatomical regions of the canine brain. The receptoractive peptides studied were methionine enkephalin, alpha-neo-endorphin, dynorphin 1-8, methionine enkephalin-Arg-Phe, and leucine enkephalin. These peptides derive from two larger precursors: proenkephalin A, which contains methionine enkephalin, leucine enkephalin, methionine enkephalin-Arg-Phe; and proenkephalin B, which contains alpha-neo-endorphin and dynorphin 1-8. Receptoractive peptides were measured in the peptide-rich fraction derived from homogenates of canine hypothalamus, pituitary, caudate nucleus, amygdala, hippocampus, mid-brain, thalamus, pons-medulla, and cortex.     

Determination of Triazines and N-Methylcarbamate Pesticides in Water by High-Performance Liquid Chromatography-Diode Array Detection

A rapid and selective method for the simultaneous determination of triazine herbicides (atrazine, its degradation product desethylatrazine, simazine, prometryn, terbutryn) and N-methylcarbamate insecticides (propoxur, carbaryl and methiocarb) in surface water has been developed. A 0.5 L of the water sample was preconcentrated by passage through a 1 g C₁₈ solid-phase extraction cartridge. The retained compounds were eluted with 5 mL of methanol from the cartridge. The pesticides were separated and quantified by reversed-phase high-performance liquid chromatography with UV diode-array detection. Analytical separation was performed using a concave gradient elution with acetonitrile and water on a C₁₈ column. Prometryn and terbutryn were determined at 240 nm; propoxur, methiocarb at 204 nm and the others at 220 nm. Recoveries varied from 85 to 102% over concentrations at 0.025 and 0.2 µg L^?1. The limits of detection for the compounds investigated are in the range of 0.005-0.012 µg L^?1.     

Combined Liquid Chromatography-Mas Spectrometry for Microscale Structural Studies of Carbohydrates

Abstract

Combined HPLC-thermospray was spectrometry of mono-and disaccharides, 1-0-methylglycosides and 0-permethy mono - through tetrasaccharicdes has been studied to asess the potential role of thermospray ionization for microscale structural studies of saccharides and glycoconjugates, and for high sensitivity detection of liquid chromatographic effluents. Using NH₄CO₂H as evaluant for reversed-phase HPLC, abundant MNH₄ ⁴ ions are formed form monosaccharides and mono-and per-methylated saccharides, and are suitable formonitoring sub-nanogram constituents in HPLC effluents. Detection of 100 pg (0.5pmole) of 1-0-methylhexopy-ranosides with signal/noise> 10 is demonstrated.     

Determination of Cefaclor in Human Plasma by Reversed-Phase High-Performance Liquid Chromatography with UV Detection and Its Application to the Bioequivalence Studies

Abstract

A selective and sensitive reversed-phase high-performance liquid chromatography method was developed and validated for quantitation of cefaclor in human plasma using cefradine as an internal standard. Calibration curve was linear over range of 0.1–20 mg · L^?1. The intra- and inter-run relative standard deviations of the assay were less than 7%. The mean absolute recoveries determined at the concentrations of 0.3, 3.0, 8.0, and 15.0 mg · L^?1 were 69.9%, 69.9%, 77.1% and 72.0%, respectively. The analytical method established was proved to be specific, precise, sensitive, and suitable for applying in the pharmacokinetic and bioequivalence studies of cefaclor in human.     

Comparative Tryptic Peptide Analysis Of Rabbit Heavy Chain Allotypes By Hplc

Abstract

Reversed-phase, high pressure liquid chromatography (HPLC) in a modified TFA/H₂O-TFA/acetonitrile gradient has been successfully applied to the structural analysis of serologlcally defined rabbit immunoglobulln heavy chains. This mobile phase modification yields a relatively flat baseline at high UV sensitivity. Approximately 40 distinct tryptic pep-tides were resolved from each heavy chain, representing the V_Ha⁺ (a), a2, and a3) and V_Ha^? (y33,30 and y33,^?) immunoglobulin allotypes. About 30 peptides were shown to be derived from the Fc region (C_H2 and C_H3), and 8–10 peptides from the Fd region (V_H1 and C_H1). Seven Fd peptides were shared by all V_Ha⁺ and V_Ha^? heavy chains. The al and a2 digests each displayed one allotype-specific peptide, whereas no allotype-specific peptides were observed for the a3 heavy chain. No differences were detected between the y33,30 and y33,^? peptides; however, both expressed a common y-specific pep-tide. Amino acid analysis of purified al-specific and y-specific peptides Indicate that the two peptides are very similar in composition to the predicted first N-terminal tryptic peptides of V_Hal and V_Ha^? heavy chains, respectively.     

Molecular Modeling for Mobile Phase Optimization in RP-HPLC

Abstract

The applicability of molecular parameters calculated on the bases of molecular mechanics have been investigated for the prediction of reversed-phase retention behavior of structurally unrelated series of drug molecules. Non-polar, non-polar unsaturated and polar surface areas, surface energies, dipole moments, van der Waals radii and hydrophobicity values expressed by the logarithm of the octanol/water partition coefficients have been calculated from the molecular structure. The reversed-phase retention behavior was described by the slope and the intercept of the straight lines obtained by plotting the log k¹ values against the acetonitrile concentration of the mobile phase. The acetonitrile concentration (OP%₀) which was needed for the log k¹ = 0 retention was also calculated from the slope and intercept values. Step-wise linear regression analyses have been applied for revealing the correlations between the investigated parameters. The slope values could be described by the difference of the non-polar and non-polar accessible surface areas or by the total surface energy values and the van der Waals radii. The intercept values could be described by the hydrophobicity parameter, the slope and the reciprocal values of dipole moment. The acetonitrile concentration for the log k'=O retention (OP%₀) could have been calculated from the hydrophobicity and the non-polar unsaturated surface area values of the investigated compounds.