Simultaneous assay of platelet and plasma catecholamines by HPLC with coulometric detection

Summary Platelets and monoaminergic neurons share many morphological, biochemical, and pharmacological properties. In addition to its similarities to serotonergic nerve endings, the platelet has also been considered a good model for the study of the noradrenergic function, since it can selectively take up, store, and release norepinephrine (NE).In this study platelet and plasma levels of free catecholamines (CA) in 20 healthy subjects have been determined by HPLC with a reversed phase (C₁₈) ionpairing system equipped with a coulometric detector. Direct correlation was observed between platelet and plasma levels of NE. A positive correlation was also found between the age of the sujects and both platelet and plasma NE levels.While showing that the peripheral noradrenergic hyperactivity of the elderly is also reflected in platelet NE content, this study also demonstrates that simultaneous assay of platelet and plasma CAs can be a useful tool for an integrated and more complete evaluation of sympathetic nervous system activity.     

Determination of highly polar catecholamine with liquid chromatography–tandem mass spectrometry using weak cation-exchange stationary phase to increase retention time

This paper reports method development and validation work to determine highly polar bases, catecholamine compounds, using weak cation-exchange liquid chromatography of low ionic strength mobile phase with electrospray tandem mass spectrometry. Catecholamine compounds, such as epinephrine and norepinephrine, well-known biomarkers to diagnose hypertension disease, spiked in saline solutions are purified with solid phase extraction (SPE) using alumina powders. The extracts are loaded into a weak cation-exchange liquid chromatographic column via an injection loop and analyzed with electrospray-mass spectrometer. The de-salted extracts contain only small amounts of electrolytes to avoid saturating weak cation-exchange sites in the stationary phase with sodium ions. Using carefully selected mobile-phase solvents with optimized compositions (acetonitrile and water 10:90 v/v) and with dilute acid additives (acetic acid 0.1% v/v), we are able to elute catecholamine at sufficient retention times to avoid co-elution of saline matrix residues while maintaining adequate electrospray ionization efficiency of these compounds. Using epinephrine and norepinephrine standards, these methods are validated at the range of 5 to 500 ng mL^− 1. The measurement accuracy and precision of using epinephrine standards are within 12% and 5.3% respectively, whereas the accuracy and precision are within 6.0% and 4.2% respectively using epinephrine standards.The detection limits of epinephrine and norepinephrine are 0.10 ng mL^− 1 and 0.45 ng mL^− 1 respectively. The recovery percentages of our solid phase extraction methods using alumina powders are higher than 74%. When the validated calibration curves are used to determine epinephrine and norepinephrine in rat blood dialysates, the determination errors of accuracy and precision are both within 4%, while the determination errors are within 3% in rat blood plasma samples.     

Effects of stationary-phase polarity on retention in reversed bonded phase HPLC columns

Summary Variations in retention and selectivity have been studied in cyano, phenyl and octyl reversed bonded phase HPLC columns. The retention of toluene, phenol, aniline and nitrobenzene in these columns has been measured using binary mixtures of water and methanol, acetonitrile or tetrahydrofuran mobile phases in order to determine the relative contributions of proton donor-proton acceptor and dipole-dipole interactions in the retention process. Retention and selectivity in these columns was correlated with polar group selectivities of mobile phase organic modifiers and the polarity of the bonded stationary phases. In spite of the prominent role of bonded phase volume and residual silanols in the retention process, each column exhibited some unique selectivities when used with different organic modifiers.     

Optimizing separations in reversed-phase liquid chromatography by varying pH and solvent composition

Summary An interpretive optimization procedure in which pH can be one of the variables is presented with the emphasis on optimizing separations. When varying the pH in reversed-phase liquid chromatography the retention of ionogenic solutes will change. Thus, the selectivity between ionogenic and neutral solutes or between ionogenic solutes mutually can be optimized. However, pH also greatly affects the efficiency (plate count) and peak shape (asymmetry). Optimum selectivity (i.e. large differences in retention times) may be observed under conditions where peaks are broad and asymmetrical. Thus, it is essential to simultaneously consider retention, peak width and peak shape and their effects on separation (effective resolution) in pH-optimization studies. A procedure in which this is done is presented and applied to optimizing the separation of a synthetic mixture of selected pharmaceuticals. After initial experiments to establish the parameter space (boundaries for pH and binary methanol — water composition), twelve experiments are performed according to a 3×4 experimental design. At each loaction the retention, peak height, peak area and peak symmetry are recorded for each solute. These data are then used to build models for each of the four characteristics and for each solute. From this set of models the response surface, describing the quality of separation as a function of pH and composition, can be calculated. A variety of optimization criteria (quantifying quality of separation) can be used. The optimum corresponds to the highest point on the response surface.     

HPLC assay of enzymatic activities

Summary Instead of traditional methods, high-performance liquid chromatography can be used for the determination of enzymatic activities. Assays can be performed once the reaction mixture have been injected in the HPLC apparatus. The elution of the samples is carried out with suitable buffers, and the product(s) formed and left oven substrate(s) are quantitatively determined by means of an internal standard. HPLC assay is rapid, selective and comparable with traditional methods. Enzymatic activity can be easily measured in international units and the whole assay performed within 10–20 minutes. The results obtained for the urokinase assay are provided as a detailed example in the text     

Solid phase extraction and direct HPLC separation of bilirubin and its conjugates in plasma

Summary A semi-automatic reversed-phase HPLC system is described for the direct determination of conjugated and unconjugated bilirubin in human plasma. An Advanced Automated Sample Processor (AASP) is used for sample preparation, and for controlling sample injection and elution. The method is highly sensitive, requiring only 10–50 μl of plasma per assay.     

Separation of urea and other polar compounds by HPLC

Summary Urea, glycolic and aminoxyacetic acid amides are the polar metabolites of 2-acetyl-3-phenyl-tetrahydro-1,2,4-oxadiazin-5-one (RGH 4615). They cannot be separated on octadecyl-, cyanosilyl- or amino-phase columns, but silica, used with C₃-C₄ alcohol and water mixtures as the eluent permits their separation. Besides refractive index detection and on-line radioactivity measurement the metabolic formation of¹⁴C-urea was proved by enzymatic cleavage into¹⁴CO₂.     

Liquid chromatographic retention of homologous alkanes in the two states of C18-bonded reversed phase silicas

Summary LiChrosorb Si100 densely grafted with octadecylmonofunctional reagents and the similar commerical LiChrosorb RP 18 have been studied in RP-HPLC, with water-methanol mobile phases at different temperatures. They exhibit a phase transition revealing two different states of bonded film as we have previously shown on densely grafted C18 or C22 macroporous silicas.The measurement of the capacity factors of the alkane homologous series indicated a discontinuity in the plot (logK, N) at a critical number whose value is dependent on temperature. Two different forms of these curves can be observed above and below the transition, revealing the influence of bonded film state on the retention mechanism.     

Determination of void/dead volume of liquid chromatographic columns containing β-cyclodextrin as the stationary phase. Part II. A new graphical method

Summary A new graphical method is proposed for the determination of the dead/void volume of liquid chromatographic columns with -cyclodextrin stationary phase. Two different approaches are presented which lead to very similar dead volume values for the cyclodextrin columns. The validity of the proposed method is discussed on the basis of column porosity values, as well as the resulting linear relationship between the logarithm of the capacity factor and the number of carbon atoms in the n-alkanol homologs. The method was applied to study the influence of various experimental parameters on the dead volume of cyclodextrin columns.     

A weak cation-exchange phase for the separation of biogenic amines by open tubular liquid chromatography

Summary The preparation and performance of a weak cation-exchange stationary phase for Open Tubular Liquid Chromatography (OT-LC) was investigated. The stationary phase was prepared in 5.4 μm I.D. fused silica capillaries byin situ photopolymerization of a mixture of silicon acrylate and acrylic acid. The influence of pH, counter ion concentration and organic modifier concentration of the mobile phase on the retention was studied with catecholamines as test solutes using LIF detection. Other biological amines like amino acids, small peptides and nucleic acid derivatives could be separated on this stationary phase as well. The kinetic performance of the stationary phase was studied with several cations and neutral solutes.     

Monolithic capillary columns synthesized from a single phosphate-containing dimethacrylate monomer for cation-exchange chromatography of peptides and proteins

Monoliths containing phosphoric acid functional groups were synthesized from only one monomer, bis[2-(methacryloyloxy)ethyl] phosphate (BMEP), in 75-μm i.d. UV transparent fused-silica capillaries by photo-initiated polymerization for cation exchange chromatography of peptides and proteins. Various synthetic conditions, including porogen solvents, monomer concentration, and polymerization time, were studied. The hydrophobicities of the resulting monoliths were evaluated using propyl paraben under reversed-phase conditions and synthetic peptides under ion-exchange conditions. These monoliths exhibited low hydrophobicities and relatively low porosities due to their highly cross-linked structures. A dynamic binding capacity (lysozyme) of 73 mg/mL of column volume was measured using the best performing monolith. Synthetic peptides were eluted in approximately 30 min without addition of acetonitrile to the mobile phase, yielding a peak capacity of 28. Efficiencies of 52,900 plates/m for peptides and 71,000 plates/m for proteins were obtained under isocratic conditions. The effects of separation conditions, i.e., mobile phase pH and salt gradient rate, were studied. Good run-to-run reproducibility was achieved with a relative standard deviation (RSD) less than 1.5% for retention times of proteins. The column-to-column retention time reproducibility for peptides was less than 3.5% RSD. A monolithic column was used to follow the deamidation of ribonuclease A. The kinetics of deamidation were founded to be first order with a half life of 195 h. A cytochrome C digest was also separated using a linear gradient of sodium chloride.     

Chromatographic classification of commercially available reverse-phase HPLC columns

Summary The ever-increasing number of commercially available reversed phases with which the analyst is confronted can cause problems in column selection. These and the non-standard test procedures used by the column manufacturers and packing companies cause further confusion. In order to independently compare and contrast a range of well established C18 stationary phases, we have performed a modified column characterization approach, based on Tanaka's methodology, on thirty commonly used phases in our laboratory. These results have been evaluated and presented in various formats as Principal Component Analysis, Cluster Analysis, Tabular Format and Radar Plots in order to assist in observing similarities and differences between phases. The results indicate that, while no two phases are exactly the same (with the exception of “me-to” clones), it is possible to characterize phases into different classes based on their chromatographic performance against selected test probes. The paper illustrates the similarities and differences between these phases. The column characterization described in the paper can form the basis of a rational column selection protocol by either careful matching of the appropriate column characteristics to the analyte's physico-chemical properties or by a systematic evaluation of columns from our various categories.     

反相HPLC同时测定茄尼醇和茄尼溴的含量

茄尼醇(solanesol;(all-E)-2,6,10,14,18,22,26,30-hexatriacontanonaen-1-ol,3,7,11,15,19,23,27,31,35-nonamethyl),白色或淡黄色固体,四倍半萜烯醇,是茄科植物烟草Nicotiana tabacum L·中含有的萜类有效成分[1],具有抗菌、消炎、抗溃疡和治疗心血管疾病等作用;可用于合成抗     

Analysis of lemon and bergamot essential oils by HPLC with microbore columns

Summary Some citrus essential oils were analyzed by HPLC with both microbore and standard columns in reversed and normal phase. Volatile and non-volatile fraction were investigated. In the non-volatile fraction some coumarins have been identified. Fractions are detected spectrophotometrically at 220 nm and 320 nm before and after evaporation of samples. Some components were also identified by LC/MS.     

Study of the separation of lactose,lactulose and galactose by liquid chromatography using cationic ion-exchange resin columns

Summary A strong acid cation-exchange resin, Dowex AG50W-X8 in potassium, calcium or sodium form, with water as eluent, has been used to separate three sugars (lactose, lactulose and galactose). The linearity of lactulose equilibrium isotherms was verified. Thermodynamic and kinetic parameters were thus determined by the moment method. Evaluation of the parameters revealed in this work can be considered as the first step in scaling up the process towards industrial use. On the other hand, the equilibrium stage model, which takes mass transfer resistance into account, is shown to provide a fair representation of the system’s behaviour.     

Simple and rugged SPE method for the determination of tetracycline antibiotics in serum by HPLC using a volatile mobile phase

Summary A simple and rugged SPE method for the determination of tetracycline (TC), minocycline (MC) and demeclocycline (DCC) in porcine serum by high performance liquid chromatography (HPLC) was developed. The spiked serum sample was pretreated with 2% phosphoric acid followed by a simple and rugged solid-phase extraction procedure using the Oasis^TM HLB extraction cartridges. High and reproducible recoveries were obtained even though the cartridges were run dry. The extracted sample analytes were injected onto a Waters SymmetryShield^TM RP₈ column. The mobile phase was a simple volatile solution containing 0.1% TFA, 2% methanol and 7% acetonitrile in Water. The antibiotics were detected at 350 nm. The calibration curves were linear from 2.0 to 25.0 μg mL⁻¹ of TC and MC with DCC as the internal standard at a concentration of 25.0 μg mL⁻¹. For six replicate analyses, the average recoveries of TC and MC from porcine serum sample fortified at the level of 2.5 μg mL⁻¹ were 96.1% with 1.3% RSD and 101% with 0.54% RSD; at level of 0.5 μg mL⁻¹ the average recoveries were 88% with 1.6% RSD and 97.8% with 1.4% RSD.     

A specific sensitive HPLC method for determination of plasma dopamine

Summary We describe here a sensitive, selective and rapid method to quantitate plasma catecholamines, especially dopamine, using high-performance liquid chromatography with electrochemical detection. This method requires a 10-minute run time and has a threshold for detection of 2 picograms, (10pg/ml).A number of commonly employed mobile phases for catecholamine analysis have been tested and have failed to detect dopamine in biological samples. Neither acetonitrile (3–7%) or methanol, (5–8%) in the mobile phase has produced consistently interpretable data either due to inability to detect or interference from co-eluting substances. Optimal detection was achieved with a mobile phase containing sodium acetate (6.8g), citric acid (5.9g), EDTA (48mg), di-n-butylamine (270l), Na-1-octane sulfate (850mg), methanol (100 ml) (amounts refer to 1 liter aqueous solution) (pH 4.3). The mobile phase was passed through a Waters 5 resolve C₁₈ column using a Waters 590 pump and m460 electrochemical detector and 740 data module, Flow rate was 0.9ml/min. Using this method, normal values in human and swine left ventricular myocardium and human and swine plasma have been established for norepinephrine, epinephrine, and dopamine.     

Silanol group effect in C18 bonded phases in HPLC

Summary If any residual (free) silanol groups remain at the surface of silica gel after bonding treatment, they may affect the retention of solutes since the dissociated groups (SiO^–) will attract cations. The silanol group effect on the retention of cationic solutes will increase with increasing pH of the mobile phase but the effect will decrease with increasing hydrophobic-ion concentration at the C₁₈ surface because such ions can mask the residual silanol groups. A method for the separation of metal complexes with 2-(5-bromo-2-pyridylazo)-diethylaminephenol (5-Br-PADAP) has been developed. The hydrophobic ion in the MeOH/H₂O mobile phase was tetrabutylammonium (TBA).     

HPLC study of the physicochemical characteristics of reverse-osmosis separation on a polyamide membrane material

Summary The fundamental characteristics of reverse osmosis on a polymer membrane have been correlated with HPLC experimental conditions by using the membrane material as a column packing. Twelve formulas were used to calculate the physicochemical characteristics of the reverse-osmosis separation process and it was found that these characteristics can be determined on the basis of retention (t _R, V_R) and partition coefficient (K) of the solute in HPLC. It seems that HPLC is an effective tool for studying the physicochemical nature of reverse-osmosis separations and the characteristics of the polymer membrane.