Richard A. Hartwick

The performance of microparticle chemically-bonded anion-exchange resins in the analysis of nucleotides.

The performance of microparticles in the ion-exchange chromatographic separation of nucleotides was investigated. A microparticle chemically-bonded strong anion-exchange column packing was used for the analysis of the 5-mono-, di- and -triphosphate nucleotides of adenine, guanine, hypoxanthine, xanthine, cytosine, uracil and thymine. It was found that excellent resolution of the majority of the nucleotides was obtained at ambient temperatures. The packings were stable and the sample capacity was greater than with pellicular resins. The retention times, peak shapes, heights and areas were highly reproducible and there was good linearity of response. Use of this column packing in biochemical studies is reported.

Identification and quantitation of nucleosides, bases and other UV-absorbing compounds in serum, using reversed-phase high-performance liquid chromatography : I. Chromatographic methodology

A comprehensive investigation of the high-performance liquid chromatographic separation of nucleosides, their bases and other low-molecular-weight UV-absorbing compounds that might be found in serum is reported. A buffer-methanol gradient was used in conjunction with chemically bonded, microparticulate columns to separate many of the biologically important compounds under study in minimal time with maximal resolution. Retention data, absorbance ratios (280/254 nm) and fluorescence responses are reported for 86 nucleosides, bases, nucleotides and other UV-absorbing compounds commonly encountered in biological studies.

Evaluation of microparticle chemically bonded reversed-phase packings in the high-pressure liquid chromatographic analysis of nucleosides and their bases.

The reversed-phase partition mode of high-pressure liquid chromatography was used for the analysis of seven of the naturally occurring nucleosides and their bases. With microparticle chemically bonded packings, nucleosides and their bases can be quantitatively determined in the presence of nucleotides in 30 min with high sensitivity, accuracy, and reproducibility. Peaks in chromatograms of cell extracts were identified by absorbance ratios and enzymatic peak shift methods. Applications of this technique to biochemical studies are reported.

Identification and quantitation of nucleosides, bases and other UV-absorbing compounds in serum, using reversed-phase high-performance liquid chromatography : II. Evaluation of human era

Abstract The reversed-phase mode of high-performance liquid chromatography was used to investigate the profiles of low-molecular-weight, UV-absorbing compounds in human serum. Identification techniques are described which allow for the identification of picomole amounts of the nucleosides, bases and other compounds in several microliters of serum ultrafiltrate. The sera from 31 normal subjects (17 males, 14 females) showed very consistent profiles. A total of 12 compounds were identified and quantified in normal serum. The analysis of sera from over 150 patients with various types of neoplasia and other diseases showed serum profiles significantly different from normal profile.

Use of UV scanning techniques in the identification of serum constituents separated by high-performance liquid chromatography

Serum constituents separated by the reversed-phase partition mode of high-performance liquid chromatography were identified by using a stopped-flow UV scanning technique. This method of peak identification eliminates post-chromatographic sample handling. Results from UV scanning were correlated with those obtained from absorbance ratios of reference compounds and the enzymatic peak-shift technique.

Sample Preparation Techniques Prior to HPLC Analysis of Serum Nucleosides and Their Bases

Abstract Techniques for sample preparation and handling of serum prior to analysis for nucleoside and bases were evaluated. Efficiency data are reported for the trichloracetic acid (TCA), ammonium sulfate, ultrafiltration and pre-column concentration sample prep-atation techniques. Maximum recoveries for most of the compounds were obtained using the ultrafiltration, ammonium sulfate and pre-column concentration techniques. Poorer recovery was observed using the TCA/Freon-amine methods. Theophylline and tryptophan exhibited pH dependent recoveries using the ultrafiltration, ammonium sulfate and pre-column techniques.

Selective analysis for adenosine using reversed-phase high-pressure liquid chromatography.

A high-pressure liquid chromatographic procedure for the selective determination of adenosine in the presence of other nucleic acid components is reported. Reversed-phase microparticle columns and an isocratic elution mode of dilute potassium dihydrophosphate and anhydrous methanol were used. The analysis is specific for adenosine and is achieved in less than 10 min. An example of the use of this analysis in a biomedical study is reported.

High performance liquid chromatographic determination of serum UV profiles of normal subjects and patients with breast cancer and benign fibrocystic changes.

High performance liquid chromatography (HPLC) was used to determine the UV profiles of serum samples taken postoperatively from 22 patients with histologically documented breast cancer, 8 patients with benign breast fibrocystic changes and 10 normal subjects. The analyses were performed on coded serum samples and after they were completed, the code was broken and the results correlated with the clinical data. Only one ml of serum was required for the HPLC analysis and identification. Detection limits for the nucleosides and bases were in the 10--20 pmol range and the injection volume of the deproteinated serum was 75 mul. The UV profiles of the normal subjects were very reproducible and similar to those of the patients with benign fibrocystic changes. The profiles of some of the cancer patients were distinctly different from the two other groups, 1-methylinosine and N2-methylguanosine, which were not detected in sera from normal subjects and patients with benign fibrocystic changes, were found in 45.5% and 22.7% of the cancer patients, respectively. Patients with the metastatic disease also showed elevated levels of guanosine and uridine. Only one false positive was found in the normal population. At present, it is not clear whether this indicates a subclinical manifestation of the disease and it must await further follow-up.

The use of High Pressure Liquid Chromatography in Clinical Chemistry and Biomedical Research

Publisher Summary High pressure, or high performance, liquid chromatography (HPLC) has potential for becoming one of the most powerful tools in the clinical laboratory. In clinical chemistry, qualitative and quantitative analyses of physiological fluids, solid wastes, and body tissues must be carried out rapidly and efficiently. If the results are to be used by physicians to detect and treat diseases as well as to monitor chemotherapy, the results must be reliable and promptly available. With HPLC, excellent resolution of closely related compounds that are polar, ionic, thermally labile, or nonvolatile can be carried out. This is most important in clinical work, because most of the drugs and their metabolites as well as many biologically active endogenous compounds fall into one of these categories. The separations are highly reproducible, and very low limits of detection are obtainable. The analyses are quantitative, and with most detectors, the samples are not destroyed. An important feature of HPLC is the ability to detect many compounds in one assay; thus concentrations of drugs and their metabolites or several drugs can be determined at one time.

High-performance liquid chromatographic assay for acid and alkaline phosphatase in serum

High-performance liquid chromatography was used to assay serum acid and alkaline phosphatase. Samples were incubated with adenosine-5-monophosphoric acid (AMP) in a buffer of required pH, 5-nucleotidase was inhibited with Ni2+ ions, and the phosphatase activity was determined by measuring the concentration of the reaction product, adenosine. The analysis time, after the incubation is terminated, is short (7 min), and the assay is quantitative and reproducible. Complete separation of the reaction product from the substrate and the naturally occurring serum constituents and the high sensitivity of the ultraviolet detection system eliminate some of the problems commonly encountered in spectrophotometric assays.