P. C. Lu

Excretion pattern investigation of urinary normal and modified nucleosides of breast cancer patients by RP‐HPLC and factor analysis method

Modified nucleosides, formed post-transcriptionally in RNA by a number of modification enzymes, are excreted in abnormal levels in the urine of patients with malignant tumors. To test their usefulness as tumor markers, and to compare them with the conventional tumor markers, a reversed-phase high-performance liquid chromatographic (RP-HPLC) method and a factor analysis method have been used to study the excretion pattern of nucleosides of breast cancer patients. A clear cut differentiation of the breast cancer group and the healthy individuals in two clusters without overlapping was obtained.

Analysis of normal and modified nucleosides in urine by capillary electrophoresisa)

SummaryModified nucleosides excreted in urine have been studied as potential diagnostic markers for cancer and AIDS, and as indicators for the whole-body turnover of RNA. Until now, reversed-phase (RP) HPLC and, to some extent, immunoassays are the preferred analytical methods for urinary nucleosides. A new capillary electrophoretic method for the analysis of normal and modified nucleosides in urine has been developed and optimized in our laboratory. The separation of nucleosides extracted from normal human urine on phenyl boronic acid affinity chromatography columns was performed in uncoated 565 mm (500 mm to detection window) × 50 μm i.d. capillary tubing using a 300 mM SDS—25 mM borate—50 mM phosphate buffer (pH 6.7), a 45-s load, a voltage of 7.5 kV (41 μA) and UV detection at 260 and 210 nm. The average recovery of the nucleosides was 91 %. The calibration curves were linear over all physiological and pathophysiological concentration ranges and the limits of detection were at micromolar levels. Reproducibility of migration times were better than 1 % (coefficient of variation,CV), and the reproducibilities of the determined concentrations were better than 5 % for standards and 6–15 % for extracted urine. The developed method was used to quantify 15 normal and modified nucleosides in 25 normal urines to establish reference ranges. The analysis time was less than 45 min.

Effect of inorganic salt concentration on the retention value in reversed phase ion-pair liquid chromatography

SummaryThermodynamic and numerical treatments have been combined with Gouy-Chapman theory were to show that the effect of the salt concentration on the capacity factor in reversed-phase ion-pair liquid chromatography (RP-IPC) can be expressed as lnk′=A+blnCs. The parameter B is related to the charge number of the solute, and the parameter A is determined by both electrostatic and nonelectrostatic interaction, and can the linearly correlated with logarithm of the capacity factor in RP-HPLC for the solute with one negative charge. It also been observed that the electrostatic interaction is reduced with increasing salt concentration in RP-IPC, and this effect is more remarkable for solutes with more than the one negative charge of sulphonic acids.

Role of solute hydrophobicity and charges in reversed-phase ion-pair liquid chromatography

SummaryThe Logarithm of capacity factor in RP-HPLC and solute charges have been selected as descriptors of solute hydrophobicity and electrostatic interaction respectively. It has been observed that the logarithm of capacity factors (ln kip′) for a solute with one, two and three negative charges in RP-IPC can be well correlated with the logarithms of capacity factors (ln kRp′) in RP-HPLC and solute charges (ne). Both the solute hydrophobicity and solute charges with opposite sign to those of the ion-pair reagent made a positive contribution to retention in RP-IPC. The logarithm of capacity factor linearly decreases with organic modifier concentration (Cb) and the logarithm of the inorganic salt concentration (lnCs). The intercept (ln kip′, A) and slope (cip,B) values of a linear relationship between ln kRp′ vs. Cb and ln kip′ vs. ln C5 also can be well correlated with ln kRp′ and ne. The solute hydrophobicity and solute charges also made a positive contribution to the parameters ln kipw and A, but a negative contribution to parameter cip. The parameter B, however is mainly determined by the solute charges.

Study of normal and modified nucleosides in serum by RP-HPLC

SummaryModified nucleosides derived predominantly from transfer ribonucleic acid (tRNA) have been studied as possible tumor markers. In this paper a reversed-phase high-performance liquid chromatographic (RP-HPLC) method has been applied to study 15 normal and modified nucleosides in serum. The nucleoside levels in normal human serum were established, and the concentrations of 15 nucleosides in serum from 19 cancer patients were determined. It was found that the concentrations of modified nucleosides were significantly higher in patient sera. Based on 15 nucleoside concentrations in serum, factor analysis could classify correctly 90% of cancer patients from the normal humans. Further work showed that urine was slightly better than serum when determining nucleosides as biological marker candidates. More work is ongoing to determine the clinical usefulness of modified nucleosides as tumor markers.

PREDICTION OF RETENTION OF O-ETHYL, O-ARYL AND N-ISOPROPYL PHOSPHOROAMIDOTHIOATES IN RP-HPLC FROM MOLECULAR STRUCTURE PARAMETERS

SummaryMulti-variable regression analysis between lnkw, c (in retention equation lnk′=lnkw+cCb) and molecular structure parameters, including hydrophobicity, electric effect, field effect and position-specific effect constant, was carried out with O-ethyl, O-aryl and N-isopropyl phosphoroamidothioates as test solutes. With these quantitative relationships, the retention behaviour of these solutes for different mobile phase compositions was predicted. The results showed that there are only 26.7% of total, experimentally measured, capacity factors with relative deviations larger than 5% and only 2.2% with deviations larger than 10%, which means that it is possible to apply the method reported to predict retention values for qualitative purposes for different mobile phase compositions.

Determination of iodine anion in dried kelp and iodized throat tablets by reversed-phase ion-pair liquid chromatography with direct UV detection

SummaryIn this paper, the amount of iodine anion in dried kelp and iodized throat tablets was determined by reversed-phase ion-pair chromatography. A 300×4 mm I.D. columns packed with μ-Bondapak-C18 (5 μm) was used and distilled water containing 10 mmol/l trimethylphenyl ammonium bromide was used as the eluent. UV detection at 231 nm was selected to monitor the iodine anion.

Classification of structurally related compounds from Astragalus extract by correlation of the logk(w) and S

SummaryThe linear relationship between logkw andS derived from molecular interactions and statistical thermodynamics was investigated by four series of different polar probe solutes. For each series of similar polar solutes, structurally related compounds with similar dipolarity/polarizability and hydrogen bonding energy, a linear relationship between logkw andS was obtained. The more similar the solutes, the greater were the regression coefficients obtained. For two series of solutes with different strong polar groups resulting in different dipolarity/polarizability and hydrogen bonding energy, two parallel lines were observed in the logkw-S plots. Each line represented one series of compounds and the distance between them indicated the difference in the dipolarity/polarizability and hydrogen bonding energy. Based on the parallel lines implying information on structurally related compounds in the logkw-S plot, a method of classification of structurally related compounds was put forward and the linear logkw-S correlation for unknown components in nonaqueous RP-HPLC analysis ofAstragalus extract with isopropanol-methanol mobile phase was studied. Two nearly parallel lines were obtained in the logkw-S plot and two series of structurally related compounds were classified in this way.

Retention behavior of dipeptide isomers in reversed-phase high-performance liquid chromatography

SummaryThe effects of eluent pH and organic modifier concentration on the capacity factor (k′) and selectivity of dipeptide isomers were investigated. It has been observed that the variation in the logarithm of the capacity factor of the dipeptide isomers is linearly dependent on the organic modifier concentration (Cb), however, the selectivity is almost independent of it. Both capacity factor and selectivity were seriously affected by the pH of the eluent. Both the capacity factor and the intercept of the ln k′ vs. Cb plot increased with increasing van der Waals volume of the non-polar amino acid subunit of the dipeptides.