Simple direct injection high-performance liquid chromatographic method to determine quinidine in plasma

High-performance liquid chromatographic methods that use direct injection of plasma include column-switching procedures, modified mobile phases and small-pore modified stationary phases. By using a large-pore (300 Å) Selectosil C₁₈ column, developed for the analysis of macromolecules, we have shown that quinidine in plasma and protein solutions can be assayed accurately and rapidly by directly injecting 2 μl plasma or protein solution onto the column. Column life is not reduced, and the limit of quantitation is 0.01 μM.     

Transport properties of alveolar epithelium measured by molecular hetastarch absorption in isolated rat lungs.

To evaluate the transport properties of the alveolar epithelium, we instilled hetastarch (Het; 6%, 10 ml, 1 - 1 x 10(4) kDa) into the trachea of isolated rat lungs and then measured the molecular distribution of Het that entered the lung perfusate from the air space over 6 h. Het transport was driven by either diffusion or an oncotic gradient. Perfusate Het had a unique, bimodal molecular weight distribution, consisting of a narrow low-molecular-weight peak at 10-15 kDa (range, 5-46 kDa) and a broad high-molecular-weight band (range 46-2,000 kDa; highest at 288 kDa). We modeled the low-molecular-weight transport as (passive) restricted diffusion or osmotic flow through a small-pore system and the high-molecular-weight transport as passive transport through a large-pore system. The equivalent small-pore radius was 5.0 nm, with a distribution of 150 pores per alveolus. The equivalent large-pore radius was 17.0 nm, with a distribution of one pore per seven alveoli. The small-pore fluid conductivity (2 x 10(-5) ml. h(-1). cm(-2). mmHg(-1)) was 10-fold larger than that of the large-pore conductivity.     

Fabrication of stratified nanoporous gold for enhanced biosensing

By a dealloying/annealing/redealloying strategy, nanoporous gold (NPG) with hierarchical microstructure is fabricated for electrochemical biosensing application. The first dealloying and annealing would produce NPG/AuAg alloy composite with a large-pore NPG layer and the second dealloying would further etch the AuAg alloy part in the composite, generating a small-pore NPG layer. By using the large-pore (≈ 100 nm) layer as the glucose oxidase (GOx) container, and the small-pore (≈ 12 nm) layer as a signal producer, this novel hierarchical NPG is demonstrated to be a good support for enzyme immobilization and fabricating enzyme-based biosensors. The immobilized GOx retains ≈ 92% of the initial activity after 7 repeated use. The GOx-loaded stratified NPG biosensor can detect glucose more sensitively with a wider linear range (up to 22 mM) than normal NPG with a uniform pore size of 30-40 nm (linear range: up to 17 mM).     

Heteropore populations of bullfrog alveolar epithelium

Diffusional fluxes of a large number of hydrophilic solutes and water across bullfrog (Rana catesbeiana) alveolar epithelium were measured in the Ussing-type flux chamber. Lungs were isolated from double-pithed animals and studied as flat sheets. Radioactive solutes and water were added to the upstream reservoir, and the rate of change of downstream reservoir radioactivity was monitored. A permeability coefficient was estimated for each substance from a linear relationship between radiotracer concentration in the downstream reservoir and time. These permeability data were used to analyze the equivalent water-filled pore characteristics of the alveolar epithelial barrier. The data reveal that the alveolar epithelium is best characterized by two distinct pore populations rather than by a single homogeneous pore population. The large-pore population consists of pores with a radius of about 5 nm and occupies 4% of the available pore area. The small-pore population consists of pores with a radius of about 0.5 nm and occupies 96% of the available pore area. The number of small pores to large pores is 2.68 X 10(3). After the alveolar surface was damaged by acid, a large-pore population with a radius of about 27 nm was seen, allowing nearly free diffusion of solutes. A major implication of the presence of two populations of pores in the alveolar epithelium is that hydrostatically driven bulk water flow occurs predominantly through the large pores, while osmotically driven bulk water flow takes place predominantly through the small pores. As a result, in general, hydrostatic and osmotic gradients may not be equally effective driving forces for water flow across this tissue.     

Comparison of different high-performance liquid chromatographic systems for the purification of adrenal and gonadal steroids prior to immunoassay

The high-performance liquid chromatography of nineteen hormonal steroids with special respect to its suitability for routine purification of these steroids from crude, organic extracts of biological fluids prior to final quantitation by immunoassay has been studied. In all systems the gradient elution technique was applied. Separation of steroids has been investigated using different stationary phases chemically coated with non-polar, hydroxyl, NO₂ and CN groups. Reproducibility of retention times was studied on a stationary phase coated with hydroxyl groups (DIOL column) using different organic eluents. Coefficients of variation range from 0.76 to 8.16%. Reproducibility was shown to be unequivocally better in the gradient part than in the isocratic part of the chromatographic run. In contrast to the other steroids, 18-hydroxylated steroids were more or less unstable in certain systems studied. As to resolution and reproducibility, the DIOL column run with an n-hexane—dioxane gradient has been shown to be superior to the other systems studied.     

Simultaneous determination of guaifenesin enantiomers and ambroxol HCl using 50‐mm chiral column for a negligible environmental impact

Chiral stationary phases are conveniently used for enantiomeric separation of drugs by liquid chromatography. Consumption of large volumes of hazardous solvents is considered as a common challenge for the sustainability of this technique. To this end, a columnar chromatography has been adopted using 50‐mm‐length stationary phases. The study comprised five Phenomenex Lux cellulose‐ and amylose‐based columns for the separation of guaifenesin (GUA) enantiomers. In addition, an experimental design was used to optimize the gradient profile for the separation of racemic GUA and ambroxol HCl (AMB) binary mixture. The chromatographic method was achieved using Lux Cellulose‐1 (50 × 4.6 mm) as a chiral stationary phase and ethanol/water as a mobile phase with linear gradient elution of 20% to 70% ethanol in 6 minutes at a flow rate of 1.0 mL min^?1 and UV detection at 270 nm. Linearity ranges were found to be 50 to 1000 μg mL^?1 and 15 to 450 μg mL^?1 for each GUA enantiomer and AMB, respectively. Environmental, health and safety tool was used to assess and compare greenness of the proposed and reported methods. Short column indeed reduces the environmental impact by decreasing waste by about 60% and utilizing only 1‐mL ethanol in the mobile phase. The proposed method is a safer alternative for the simultaneous determination of drugs in their combined pharmaceutical formulation. The method has been validated and compared favorably with a reported one.     

Evaluation of stationary phases for 2-dimensional HPLC of proteins Part 1. Validation of commercial RP-columns

RP-separation with TFA-based water/acetonitrile eluents is widely used for peptides and small proteins but is well known difficult for large or membrane proteins. Especially in proteomics or other complex biological matrices reliable elution patterns are difficult to achieve. New commercial stationary phases are validated regarding long term stability, protein recovery, carry over, symmetry and selectivity using 10 different proteins with different molar weights, isoelectric points and glycosylation. It could be demonstrated that some stationary phases had poor protein elution performances. They did not elute a protein at all or with minor recovery, peak symmetries. Sometimes bad and formidable carry over effects for peak areas in the following run were observed. Selectivity in separation of different isomers or glycosylated proteins is also different. The results suggest that neither surface area nor pore diameter play an important role in the application of reversed phases for HPLC of proteins. The investigations leads one to suppose that the bonding chemistry seems to be an important aspect. Most critical fact is that some RP-phases did not elute a protein at all others only 20% of the injected protein mass, which makes the objective of an RP-chromatogram highly questionable.     

Protein unfolding during reversed-phase chromatography: I. Effect of surface properties and duration of adsorption.

Residue-level features of bovine pancreatic trypsin inhibitor (BPTI) unfolding on reversed-phase chromatography (RPC) surfaces were investigated using hydrogen-deuterium exchange labeling and NMR. A set of silica-based RPC surfaces was used to examine the influence of alkyl chain length and media pore size on adsorbed BPTI conformation. In all cases there was substantial unfolding in the adsorbed state; however, residual protection from exchange was consistently observed. Particle pore size did not influence conformation substantially for C4-alkyl modified silica; however, 120 A pore C18 media produced more hydrogen exchange than any other surface examined. In this case, the radius of curvature inside the pore approaches the size of the BPTI molecule. Generally, the pattern of hydrogen exchange protection was uniform; however, the beta-sheet region was selectively protected on the large-pore C18 media. The beta-sheet region forms a hydrophobic core that forms early when BPTI folds in solution. This suggests that partially unfolded states possessing a native-like structure play an important role in adsorption and elution in RPC. Finally, increased contact time with the surface before elution fostered unfolding and altered chromatographic behavior considerably.     

Determination of methotrexate in human urine at nanomolar levels by high-performance liquid chromatography with column switching

An high-performance liquid chromatographic method with column switching for the detection of less than 4 ng of methotrexate in the urine of oncologic nurses is described. Urine samples were purified by solid-phase extraction on silica-bonded phenyl columns, eluting impurities with ethyl acetate. After elution from the column, the analyte was concentrated ten-fold, evaporating the solvent. On a strong anion-exchange column (Nucleosil 100 SB), methotrexate was separated from the remaining interfering substances, was then switched to a reversed-phase column (LiChrospher 100 RP-18e), and finally eluted by a linear gradient in a solvent system consisting of ammonium formate buffer (pH 2.7) and acetonitrile. Absorbance was monitored at 310 nm. This method has proved to be suitable for detecting traces of methotrexate in urine in order to individualize risks and to reduce further the occupational safety hazard for hospital personnel.     

Sepsis in sheep reduces pulmonary microvascular sieving capacity

The changes in pulmonary microvascular permeability in sheep, after infusion of live Escherichia coli, were studied using estimations of the osmotic reflection coefficients (sigma) for total protein, albumin, immunoglobins (Ig) G and M and based on these estimations equivalent pore dimensions were calculated. A chronic lung lymph fistula was prepared in seven sheep. After a base-line period, left atrial pressure (Pla) was increased. E. coli (10(9) X kg body wt) were given after attaining filtration independent L/P values. The sigma's for the normal lung were calculated to 0.73 for total protein and to 0.65, 0.76, and 0.91 for albumin, IgG, and IgM, respectively. The equivalent pore radii were determined to 50 and 175 A with 35% of the filtration accounted for by the large pores. After bacterial infusion, the sigma's for total protein, albumin, IgG, and IgM decreased significantly from preseptic values to 0.58, 0.50, 0.64, and 0.83, respectively. After sepsis the small pores were 50 A and the large pores 200 A with 49% of total volume flow at maximum lymph flows occurring through the large pores. Assuming a constant small-pore population the large-pore number increased 32% after bacterial infusion. These results indicate that pulmonary microvascular permeability may have increased due to the sepsis.     

HPLC enantioseparation on cellulose tris(3,5-dimethylphenylcarbamate) as a chiral stationary phase: Influences of pore size of silica gel,coating amount,coating solvent,and column temperature on chiral discrimination

Cellulose tris(3,5-dimethylphenylcarbamate) (CDMPC) was coated on large-pore silica gels and used as a chiral stationary phase (CSP) for high-performance liquid chromatographic separation of enantiomers. The influences of pore size of silica gel, coating amount of CDMPC, coating solvent, and column temperature on chiral discrimination were investigated. CSPs prepared with a large-pore silica gel having a small surface area showed higher chiral recognition. The amount of CDMPC adsorbed on the silica gel influenced the chiral recognition of some racemates. Loading capacity of racemates increased with an increase of the amount of CDMPC supported on the silica gel, and a CSP coated with 45% CDMPC by weight can be used for both analytical and semi-preparative scale separations. The CDMPC, coated using acetone as the coating solvent, exhibited, in many cases, higher enantioselectivity than that obtained with tetrahydrofuran F as the coating solvent. © 1996 Wiley-Liss, Inc.     

Enantioseparation of racecadotril using polysaccharide‐type chiral stationary phases in polar organic mode

Enantioseparation of the antidiarrheal drug, racecadotril, was investigated by liquid chromatography using polysaccharide‐type chiral stationary phases in polar organic mode. The enantiodiscrimininating properties of 4 different chiral columns (Chiralpak AD, Chiralcel OD, Chiralpak AS, Chiralcel OJ) with 5 different solvents (methanol, ethanol, 1‐propanol, 2‐propanol, and acetonitrile) at 5 different temperatures (5–40 °C) were investigated. Apart from Chiralpak AS column the other 3 columns showed significant enantioseparation capabilities. Among the tested mobile phases, alcohol type solvents were superior over acetonitrile, and significant differences in enantioselective performance of the selector were observed depending on the type of alcohol employed. Van't Hoff analysis was used for calculation of thermodynamic parameters which revealed that enantioseparation is mainly enthalpy controlled; however, enthropic control was also observed. Enantiopure standard was used to determine the enantiomer elution order, revealing chiral selector—and mobile‐phase dependent reversal of enantiomer elution order. Using the optimized method (Chiralcel OJ stationary phase, thermostated at 10 °C, 100% methanol, flow rate: 0.6 mL/min) baseline separation of racecadotril enantiomers (resolution = 3.00 ± 0.02) was achieved, with the R‐enantiomer eluting first. The method was validated according to the ICH guidelines, and its application was tested on capsule and granules containing the racemic mixture of the drug.     

Reversed-phase HPLC enantioseparation of pantoprazole using a teicoplanin aglycone stationary phase—Determination of the enantiomer elution order using HPLC-CD analyses

A direct HPLC method was developed for the enantioseparation of pantoprazole using macrocyclic glycopeptide-based chiral stationary phases, along with various methods to determine the elution order without isolation of the individual enantiomers. In the preliminary screening, four macrocyclic glycopeptide-based chiral stationary phases containing vancomycin (Chirobiotic V), ristocetin A (Chirobiotic R), teicoplanin (Chirobiotic T), and teicoplanin-aglycone (Chirobiotic TAG) were screened in polar organic and reversed-phase mode. Best results were achieved by using Chirobiotic TAG column and a methanol-water mixture as mobile phase. Further method optimization was performed using a face-centered central composite design to achieve the highest chiral resolution. Optimized parameters, offering baseline separation (resolution = 1.91 ± 0.03) were as follows: Chirobiotic TAG stationary phase, thermostated at 10°C, mobile phase consisting of methanol/20mM ammonium acetate 60:40 v/v, and 0.6 mL/min flow rate. Enantiomer elution order was determined using HPLC hyphenated with circular dichroism (CD) spectroscopy detection. The online CD signals of the separated pantoprazole enantiomers at selected wavelengths were compared with the structurally analogous esomeprazole enantiomer. For further verification, the inline rapid, multiscan CD signals were compared with the quantum chemically calculated CD spectra. Furthermore, docking calculations were used to investigate the enantiorecognition at molecular level. The molecular docking shows that the R-enantiomer binds stronger to the chiral selector than its antipode, which is in accordance with the determined elution order on the column—S- followed by the R-isomer. Thus, combined methods, HPLC-CD and theoretical calculations, are highly efficient in predicting the elution order of enantiomers.     

High-performance reversed-phase chromatographic mapping of 2-pyridylamino derivatives of xyloglucan oligosaccharides.

Xyloglucan oligosaccharides from cotton cell walls and tamarind seeds were derivatized with 2-aminopyridine and subsequently separated by reversed-phase chromatography (r.p.c.) using an octadecylsilyl silica stationary phase and aqueous-organic eluents with 0.01% (v/v) trifluoroacetic acid. The chromatographic behavior of the 2-pyridylamino derivatives of xyloglucan oligosaccharides was examined under a wide range of elution conditions, including gradient steepness and shape, initial acetonitrile concentration in the eluent, and pore size of the r.p.c. packings. Relatively steep acetonitrile gradients resulted in poor resolution of the different xyloglucan fragments, which is believed to be the result of acetonitrile-induced conformational changes. Under these circumstances the elution order of the derivatized xyloglucan oligosaccharides was such that the smaller fragments eluted from the column before the larger ones. R.p.c. packing with a 70-A pore size necessitated relatively high acetonitrile concentration in the eluent when compared with 300-A stationary phase. The r.p.c. mapping of 2-pyridylamino derivatives of xyloglucan oligosaccharides was best achieved when both a wide-pore octadecyl-silyl silica stationary phase and a shallow gradient with consecutive linear segments of increasing acetonitrile concentration in the eluent were employed. This combination yielded rapid r.p.c. maps of the xyloglucan fragments from different sources with high separation efficiencies and concomitantly high resolution. The effects of the nature of the sugar residues in the xyloglucan oligomers and their degree of branching on r.p.c. retention and selectivity are also highlighted.     

Lipophilicity Determination of Highly Lipophilic Compounds by Liquid Chromatography

Different experimental strategies using short columns in both conventional liquid chromatography (HPLC) and ultra‐high pressure liquid chromatography (UHPLC) were evaluated to allow, for the first time with these techniques, the lipophilicity determination of compounds with log P>5. Various organic modifiers, stationary phases, and elution modes were tested on 14 rigid compounds with a CLogP between 5 and 8, and 38 compounds with log P_oct from 0 to 5. The best results in HPLC were obtained with the 20‐mm Discovery ^® RP Amide C16 stationary phase in isocratic mode using MeOH as organic modifier. To improve analysis time, the UHPLC approach was then evaluated. Consequently, a generic method was developed with a 30‐mm Acquity BEH Shield RP18 column in gradient mode using MeOH as organic modifier, allowing a fourfold gain of time compared to the HPLC method, for the highly lipophilic compounds tested. Finally, the most rapid and accurate results were obtained with a 10‐mm Hypersil^TM GOLD Javelin HTS stationary phase in UHPLC, enabling an eightfold gain of time compared to the HPLC method.     

Fractionation of human low density lipoprotein by column chromatography.

We have devised a method to fractionate low density lipoprotein (LDL) into subspecies by means of column chromatography. DEAE-agarose columns, 2.6 X 60 cm, were loaded with LDL (25-45 mg LDL protein) and eluted with a 0.045-0.13 M NaCl gradient. The LDL eluted over a volume of 900 ml. Specific portions of the eluted LDL, reapplied to a column identical with the original, reelute at about the same point. Altering the NaCl concentration of the elution fluid changed the elution volume. The cholesterol-protein ratio of the LDL subfractions was progressively lower in fractions eluting at higher NaCl concentrations. These results indicate the LDL is not a homogenous lipoprotein species but consists of subfractions which differ in at least charge and cholesterol content.     

Comparison of Lysyl-Transfer Ribonucleic Acid Species from Vegetative Cells and Spores of Bacillus subtilis by Methylated Albumin-Kieselguhr and Reversed-Phase Chromatography

Lysyl-transfer ribonucleic acid (tRNA) species from a spore-forming strain of Bacillus subtilis (168 trp2(-)) and an early blocked asporogenous mutant (spoA 12) were compared on reversed-phase and methylated albumin-kieselguhr columns. Lysyl-tRNA species from spores and the asporogenous mutant in stationary phase both exhibited altered chromatographic profiles compared to that of log-phase cells. The major peak in spore lysyl-tRNA species eluted later than that characteristic of vegetative cells, whereas the major peak of the lysyl-tRNA species from the asporogenous mutant in stationary phase eluted earlier. Although the early eluting lysyl-tRNA species was observable on methylated albumin columns, the late eluting peak was not detectable by that column technique. By using a shallower gradient on an RPC-2 column, the resolution of all lysyl-tRNA species increased. Several subspecies were revealed. The chromatographic comparisons clearly show that both the spore-forming strain and the asporogenous mutant undergo relative increases in different lysyl-tRNA species when grown to late stationary phase. No new species seem to be involved but rather altered amounts of minor species existing in log-phase cells. The experiments also demonstrate the usefulness of reversed-phase columns for such comparisons.     

High Performance Liquid Chromatographic Analysis of Cytokinins in Sorghum bicolor Leaves

High performance liquid chromatography with octadecylsilica (Bondapak C₁₈/Poracil B) column packing was used to purify and separate cytokinins in Sorghum leaf extracts. The column size was 56 × 0.21 cm i.d. By gradient elution, using acidified water with increasing amounts of methanol, the major peaks of cytokinin activity, as determined by the callus tissue bioassay. were effectively separated from large amounts of extraneous impurities. These cytokinins were further separated on a microoctadecylsilica column (μBondapak C₁₈, 30 × 0.4 cm i.d.) with a gradient of acidified water-acetonitrile. Zeatin and zeatin riboside gave distinct ultra violet absorption peaks which could be used for quantitative estimation. Biological activity corresponded to the elution of these peaks. These two cytokinins are the major cytokinins in Sorghum leaves.     

Modeling pore size distribution in cellulose rolled stationary phases

Rolled stationary phases are fabrics (i.e., nonparticulate phases) that rapidly separate proteins from salts on the basis of size exclusion. Pore size and pore size distributions in the stationary phase determine how different size molecules distribute between the stationary and mobile phases in liquid chromatography columns. The potential for size exclusion chromatography by fabrics is not initially obvious because their interlaced structures are atypical for size exclusion supports. A simple logistic model fits the pore size distribution of a rolled stationary phase when pore sizes were measured using PEG, Dextran, D2O, glucose, and NaCl probes. When the fabric is treated with cellulase enzymes, the water-accessible pores uniformly decrease and peak retention is lower. The logistic function model captures this result and enables comparison of pore size distribution curves between enzyme-treated and untreated fabrics in rolled stationary phase columns.     

Evidence for an interaction between cytosolic aldolase and the ATP-and pyrophosphate-dependent phosphofructokinases in carrot storage roots.

Immunoaffinity chromatography was employed to identify potential plant cytosolic aldolase (ALDc) binding proteins. A clarified homogenate of carrot storage root was chromatographed on a column of protein-A-Sepharose that had been covalently coupled to anti-(carrot root ALDc) immunoglobulin G. The column was washed with phosphate-buffered saline (PBS), followed by step-wise elution with increasing concentrations of NaCl in PBS. Several proteins were eluted following application of the salt gradient. Western blotting identified the major eluting proteins to be the PPi-dependent phosphofructokinase (PFP) and the cytosolic form of the ATP-dependent phosphofructokinase (PFKc), enzymes that are metabolically sequential to ALDc. The results suggest that ALDc may specifically interact with PFP and PFKc in carrots.