Polymeric Reversed-phase Column Research Articles

Determination of urinary 8-hydroxydeoxyguanosine by automated coupled-column high performance liquid chromatography: a powerful technique for assaying in vivo oxidative DNA damage in cancer patients

An automated analytical method has been developed for determination of the oxidative DNA adduct, 8-hydroxydeoxyguanosine (8OHdG) in human urine, based on coupled-column high performance liquid chromatography with electrochemical detection. Urine is concentrated on Bondelut CH by means of an automated sample processor, and the enriched sample injected on to a polymeric reversed phase column coupled in line with an electrochemical detector and a C 18 reversed phase column. By use of the electrochemical detector, a suitable retention time interval is set for collection of the fraction containing 8OHdG from the chromatography on the first column; this fraction is collected in a 2 mL loop and injected onto the C 18 column. The system is operated by an automatic valve station controlled by an integrator. The method has a large sample capacity and measures 31.1, 15.7, and 7.43 nmol 8OHdG/L urine with variation coefficients of 8, 8 and 24% within series and 8, 11 and 23% between series. Normal healthy individuals were found to excrete 14.9 ± 7.8 nmol 8OHdG/24 h, or 1.11 ± 0.62 μmol 8OHdG per mol creatinine, in their urine, whereas increased levels of 8OHdG were found in 24 h collections from a variety of cancer patients, both in samples taken before onset of oncological therapy (1.84 ± 1.12 μmol/mol creatinine, P < 0.01 versus healthy individuals) and after therapy onset (2.18 ± 1.44 μmol/mol creatinine, P < 0.001 versus healthy individuals). Moreover, mean values of 8OHdG in random urinary samples from cancer patients were significantly higher than from healthy individuals (2.42 ± 2.28 versus 1.19 ± 0.48 μmol/mol creatinine, P < 0.001), both in samples taken before therapy onset (1.91 ± 0.96, P < 0.001 versus healthy individuals) and after (2.57 ± 2.46, P < 0.001 versus healthy individuals). High levels of urinary 8OHdG were found in patients subjected to whole body irradiation, and in patients receiving chemotherapy with various cytostatic agents. The potential use of the method for detecting increased urinary 8OHdG excretion and conditions associated with increased oxidative DNA damage in humans is discussed.

Simplified clean-up for the determination of benzimidazolic fungicides by high-performance liquid chromatography with UV detection

A method was developed that allows the determination of benomyl, carbendazim (MBC), thiophanate methyl (TFM) as carbendazim and thiabendazole (TBZ) by HPLC with UV detection. After extraction and cyclization of TFM into MBC, the conversion of benomyl into MBC is carried out by absorbing the raw extract on a ready-to-use, disposable column of a macroporous Kieselghur-type material and percolating 0.1 M HCl through it. Benzimidazolic residues are partitioned into the acid solution whereas most of the co-extractives are retained on the column. The final clean-up is performed on a strong cation-exchange (SCX) cartridge. The determination of MBC and TBZ is carried out by HPLC-UV detection on a polymeric reversed-phase column eluted with a water-ecetonitrile (70 : 30). Recoveries of MBC and TBZ from pear, apple, orange, grape, kiwi, tomato and lettuce, spiked at levels of 0.22 and 0.88 mg/kg, were satisfactory (70%). The main features of the method include high selectivity towards MBC and TBZ, reduced consumption of reagents and solvents, reduced handling operations, lack of emulsions and the use of disposable items.

Determination of the aza alkyl lysophospholipid 3-methoxy-2-N,N-methyloctadecylaminopropyloxyphosphorylcholine in rat plasma by liquid chromatography—particle beam—mass spectrometry

A sensitive and specific method for the determination of the aza alkyl lysophospholipid (AALP) 3-methoxy-2-N,N-methyloctadecylaminopropyloxyphosphorylcholine (I) in rat plasma is described. The target molecule was analyzed by high-performance liquid chromatography (HPLC)-mass spectrometry (MS) after one single liquid-liquid extraction with chloroform-methanol (2:1, v/v). 1,2-Didecanoyl-sn-glycero-3-phosphocholine was used as internal standard. HPLC was carried out using a polymeric reversed-phase column; the coupling to the mass spectrometer was a particle beam (PB) interface, and the ionization method was electron impact (EI). This simple and rugged method permits the measurement of I in rat plasma in the range of 25 ng/ml-5 micrograms/ml with good accuracy and precision and is used in pharmacokinetic studies.

High performance liquid chromatographic separation of polar and non‐polar chlorophyll pigments from algae using a wide pore polymeric octadecylsilica column

AbstractA method employing a wide pore polymeric reversed phase column has been developed for the separation of most of the chlorophylls and related compounds previously described as occurring in marine microalgae. The high selectivity toward molecular shape of this kind of stationary phase has enabled compounds of very similar structure, such as chlorophylls c1, c2 and Mg‐divinylpheoporphyrin a5 monomethyl ester, and chlorophyll a and the phytol‐substituted chlorophyll c‐like pigments, which commonly coelute on monomeric bonded phases, to be resolved in a single run. Some of these pigments, formerly thought to be a single compound, have, in fact, been demonstrated to be groups of two or more. The method has been successfully applied to both algal cultures and natural sea water samples. When visible light absorbance detection was used, the method proved suitable for separation of various carotenoids.

Fast protein monitoring in fermentation broth using non-porous micropellicular reversed-phase columns

The use of a short non-porous polymeric reversed-phase column is described for the rapid monitoring of the formation of recombinant human granulocyte colony stimulating factor in fermentation broths. The described procedure is capable of completing the assay in less than 10 min. Validation of the method showed acceptable recovery, accuracy, precision and specificity.

Silica versus polymer-based stationary phases for reversed-phase high-performance liquid chromatographic analyses of rat insulin biosynthesis : A comparison of resolution and recovery

Because of the problems caused by the irreversible binding of insulins and proinsulins to several silica-based reversed-phase columns, the use of polymeric reversed-phase columns was investigated for the analysis of rat islet polypeptides involved in insulin biosynthesis. No irreversible binding of insulins and proinsulins was observed for the polymeric reversed-phase columns, probably due to the absence of silanol groups. The six polypeptides involved in insulin biosynthesis in rat islets were equally well resolved in shallow trifluoroacetic acid-acetonitrile gradients on the silica-based Nucleosil 300-5C 4 column (45°C), the polymer-based Asahipak C4P-50 (25 and 45°C), and ODP-50 columns (45°C). In shallow triethylammonium phosphate-acetonitrile gradients (25°C) satisfactory resolution of the two rat proinsulins was only obtained on the polymer-based Asahipak C4P-50 and C8P-50 columns. Increasing the separation temperature to 45°C improved the separation of the two insulins and the two proinsulins in all cases. The shifts in retention times for the individual islet polypeptides observed in relation to the increased separation temperature were found to be different for the silica C 4 and the polymer C 4 columns. Recoveries of rat islet polypeptides were comparably high from the silica- and the polymer-based C 4 columns and linear load-response curves were obtained in the microgram to picogram mass range of both columns.

Analysis of tropane alkaloids with thermospray high-performance liquid chromatography—mass spectrometry

A thermospray high-performance liquid chromatography—mass spectrometry method for analysis of hyoscyamine and scopolamine in plant cell culture samples is described. The alkaloids were separated on a polymeric reversed-phase column with an alkaline ammonium acetate buffer—acetonitrile eluent. Selected-ion recording of the protonated molecular ions was used for quantitation of the compounds. The compounds were fragmented by discharge-assisted ionization and elevated thermospray capillary temperatures or ion repeller potentials.

Use of polymeric reversed-phase columns for the characterization of polypeptides extracted from human pancreata : I. Effect of the mobile phase

The high-performance liquid chromatographic (HPLC) behaviour of two different styrene-divinyl-benzene-based reversed-phase (RP) columns was evaluated using crude acetic acid extracts from normal and diabetic human pancreata as samples. Acetic acid gradients in water and acetonitrile gradients in triethylammonium phosphate (TEAP) and trifluoroacetic acid (TFA) were used as mobile phases, and comparisons were made with a silica-based C 4 column. When two different polymeric RP columns were eluted with acetic acid gradients in water, surprisingly similar HPLC profiles of the pancreatic extracts were obtained. Elution of the polymer-based columns with acetonitrile gradients in TFA or TEAP resulted in changes in the polypeptide selectivity of these columns, in parallel with that of a silica-based C 4 column eluted under similar conditions, indicating the general usability of polymeric columns for RP-HPLC of peptides and proteins. The pronounced difference in composition between normal and diabetic samples, which also was demonstrated after size-exclusion chromatography (SEC) on a silica-based and an agarosebased high-performance SEC column, was found to be related to the different ischaemia times for the two types of pancreata.

Use of polymeric reversed-phase columns for the characterization of polypeptides extracted from human pancreata : II. Effect of the stationary phase

The potential value of eight commercial available polymer-based reversed-phase (RP) columns for peptide and protein separations was evaluated using crude acetic acid extracts of normal and diabetic human pancreata and mixtures of pure polypeptides as samples. All columns were characterized with acetic acid gradients in water as mobile phase, and different chromatographic profiles were obtained depending on the type of polymer column (bare or derivatized) and the type of ligand. Some of the columns were virtually free from effects related to the polymer skeleton whereas in others the separation was influenced by both the ligand and the polymeric backbone. Two selected polymeric RP columns were, together with a silica-based C 4 column, further characterized with acetonitrile gradients in trifluoroacetic acid (TFA), and the separation temperature was found to have a drastic effect on the separation efficiency for proteins with mol. wt. > 6000 dalton. No such effect was seen for polypeptides with mol. wt. < 6000 dalton. Mixtures of pure peptides and proteins were separated using acetic acid gradients in water, acetonitrile or isopropanol, and normally the highest efficiency was found with the use of acetonitrile as mobile phase modifier. Isopropanol was less suitable as an organic modifier. The separation of the β-lactoglobulin A- and B-chains may be used to give a rapid estimate of the chromatographic usability of polymer-based RP-columns for peptide and protein separations in acetic acid gradients in water and in acetonitrile gradients. Recoveries for insulin, proinsulin, growth hormone, ovalbumin and human serum albumin were measured for several polymer-based RP columns eluted with acetic acid gradients in water and with acetonitrile-based mobile phases. The highest recoveries of serum albumin and ovalbumin were found after elution with acetic acid gradients in water.

Selectivity of some aromatic compounds and carotenoids on C18 packings made with different endcapping procedures

Monomeric and polymeric reversed-phase (C18) column packing materials have been prepared for HPLC. Columns have been tested with aromatic hydrocarbons, acidic and basic solutes. Good separation of carotenoids can be achieved on well-endcapped phases, but at the separation of some pairs (e.g. carotenoids with ∈-and β-endgroups) nonendcapped materials give better resolution. The influence of coating structure and that of endcapping have been discussed.

Optimization of high-performance liquid chromatographic peptide separations with alternative mobile and stationary phases

Peptides are routinely separated with reversed-phase high-performance liquid chromatography using increasing concentrations of acetonitrile in the presence of trifluoroacetic acid. While these separations may be improved by adjustments of gradient slope or substitutions of different solid-phase chemistries, many mixtures would benefit from systematic optimization of mobile phase components. Tryptic digests of cytochrome c from various species were separated on Waters Delta-Pak TM C 18. The effects of varying pH as well as the concentration and type of ion-pair reagent were examined. In addition, low pH, ion-suppression/ion-pairing chromatography was inverted using a polymeric reversed-phase column at high pH with alkyl amine ion pairing. Finally, a tryptic digest of cytochrome c was resolved by ion-exchange chromatography with a strong cation-exchange high-performance liquid chromatography column. These data suggest a framework for dramatically changing the selectivity of peptide separations, leading to more satisfactory peptide mapping.

Determination of diltiazem in human whole blood and plasma by high-performance liquid chromatography using a polymeric reversed-phase column and utilizing a salting-out extraction procedure.

A rapid, simple and sensitive reversed-phase high-performance liquid chromatography method for the separation and measurement of the concentration of diltiazem in human whole blood and plasma has been developed. The method involves a simple one-step solvent extraction of the drug from biological samples followed by salting-out the organic solvent using ammonium sulfate. A 15 cm x 4.1 mm, PRP-1, pH-stable (pH 1-13) polymeric reversed-phase column was used with an isocratic elution of acetonitrile-0.01 M tetrabutylammonium hydroxide (60:40, v/v). Diltiazem was monitored at 254 nm and 0.50-0.005 a.u.f.s. The completion time for assay was less than 15 min, and the limit of detection was 10 ng/ml at a signal-to-noise ratio of 3 for a 50-microliters injection volume. Using this method, the pharmacokinetic parameters (elimination rate constant, elimination half-life and area under the curve) were calculated from a whole blood concentration versus time profile of diltiazem.

Thermodynamic comparison of monomeric and polymeric C 18 bonded phases using aqueous methanol and acetonitrile mobile phases

The results of a thermodynamic comparison of the retention of four polycyclic aromatic hydrocarbons on a monomeric and a polymeric reversed-phase column using methanol and acetonitrile are presented. The results are discussed in terms of enthalpic and relative entropic contributions to overall retention.

Comparison of bonded, polymeric and silica columns for chromatography of some penicillins

The retention of ampicillin, amoxicillin, and penicillin G was determined on ODS and cyanopropyl (CN)-bonded to silica, on an all-organic polymeric reversed-phase column (styrene-divinylbenzene) and on bare silica. Solvents tested were 0.01 M H 3PO 4 (pH 1.6)-acetonitrile and 0.002 M KH 2PO 4 (pH 4.6)-acetonitrile in the proportions 100:0 to 10:90. Retention with 0.002 M KH 2PO 4 was weaker than with 0.01 M H 3PO 4 at all proportions of acetonitrile on all packings. With 0.01 M H 3PO 4, retention of penicillins on the polymeric column decreased rapidly from 100 to 50% aqueous phase and remained low to 10% aqueous phase. On the ODS bonded column, retention was similar, except that retention of the aminopenicillins, amoxicillin and ampicillin increased at < 30% aqueous phase. On the CN-bonded column, results were similar to those on the ODS column, except that the aminopencillins were less strongly retained when using 100% 0.01 M H 3PO 4. On the bare silica, penicillin G was poorly retained at all proportions of acetonitrile, while retention of aminopenicillins increased rapidly at < 50% 0.01 M H 3PO 4. Retention of aminopenicillins at < 50%. 0.01 M H 3PO 4 was parallel on bonded and bare silica but weaker on the bonded silica. A bonded organic layer weakened but did not prevent binding of amines to silica.

Effect of the silica support of bonded reversed-phase columns on chromatography of some antibiotic compounds

Chromatographic behavior of three types of antibiotics was investigated on bonded C 18 and polymeric reversed-phase columns. With penicillins with carboxylic acid functions only, retention and separations on the two types of columns were similar. β-Lactam antibiotics with basic functions did not give as sharp peaks on the C 18 column unless a silanol blocking agent, tetramethyl ammonium chloride (TMA) was added. In 0.01 M orthophosphoric acid-acetonitrile, tetracyclines were separated on the polymeric reversed-phase columns, but not on the C 18 columns. With addition of TMA, results on C 18 and polymeric reversed-phase columns were nearly identical. Addition of an ion pair also improved separations on the C 18 columns, but not as much as TMA. Interaction with the silica support of C 18 columns was used to separate tylosin from interferences in extracts of biological materials. The results demonstrate the importance of interactions with the silica support in chromatography of basic antibiotics on C 18 packings. These interactions can be either beneficial or detrimental to separations, depending on the conditions used.

Chromatographic separation of benzo(a)pyrene isomers on nematic liquid crystal GC and polymeric reversed-phase HPLC phases

The use of two nematic liquid crystals (BABT and BPhBT) as GC stationary phases for the separation of monohydroxybenzo(a)pyrenes. as their TMS ethers, and monomethylbenzo(a)pyrenes was developed and compared with the separation of these isomers by HPLC using a polymeric ODS reversed-phase column. It was found that while HPLC and GC gave comparable separation of the hydroxy isomers, 10 out of 12 separated, better separation of the methyl isomers was obtained using HPLC. A simultaneous use of both HPLC and GC would resolve the twelve hydroxy isomers in about 70min. The results indicated that HPLC, using polymeric reversed-phase columns, is as powerful a tool as GC using nematic liquid crystal phases, for the separation of benzo(a)pyrene isomers. A discussion of the effect of solute length-to-breadth ratio on elution order is presented.