Biomedical Chromatography

doi: 10.1002/bmc.1130030301pmid: N/A

doi: 10.1002/bmc.1130030311pmid: N/A

doi: 10.1002/bmc.1130030312pmid: N/A

Bhusan, R; Mahesh, V. K.; Mallikharjun, P. V.

doi: 10.1002/bmc.1130030302pmid: 2670009

Gerhardt, Greg A.; Drebing, Carla J.; Stephen, Christopher; Freedman, Robert

doi: 10.1002/bmc.1130030303pmid: 2765691

We report a procedure for the rapid determination of unconjugated homovanillic acid (HVA) in human plasma by cartridge filtration followed by direct injection into an isocratic HPLC system coupled with dual‐electrode coulometric electrochemical detection. Sample preparation is rapid and more than 72 samples can be studied in 24 h using an automated HPLC system. The intra‐ and interassay precisions of the assay equal or exceed previously reported methods. While this methodology has been employed to study plasma free HVA, the concept of the technique may be applicable to other compounds, as well as different assay procedures, to greatly decrease sample preparation time.

Harada, Hiroshi; Ueno, Yasushi; Kamei, Masugu; Ohura, Reiko; Tanabe, Noriko; Uchida, Yasuko; Koyama, Fumihiro; Yamaguchi, Takashi

doi: 10.1002/bmc.1130030304pmid: 2504311

Fluorescein isothiocyanate (FITC)‐labelled asialotransferrin and pyridyl aminated oligosaccharides were prepared from asialotransferrin and human milk using affinity chromatography and high performance liquid chromatography (HPLC), respectively. These substances were incubated with galactosidase or sialyltransferase and then examined by lectin affinity HPLC. The elution patterns changed according to the period of incubation and amount of enzyme. This analytical method using lectin affinity HPLC with fluorescence labelled glycoprotein or oligosaccharides as the substrates has great value for detecting these enzyme under the same chromatographic conditions. In addition, differences were noted in the activity of β‐galactosidase toward oligosaccharides having the Gal β(1 → 3)GlcNAc or Gal β(1 → 4)GlcNAc structure at reducing termini.

Koel, Marlies; Nebinger, Peter

doi: 10.1002/bmc.1130030305pmid: 2475195

A HPLC method for the quantitative determination of 5‐hydroxy‐3‐indoleacetic acid (5‐HIAA) in urine is described. The method is based on ion‐pair chromatography, reversed phase (RP) column material and specific fluorimetric detection at 300 nm and 355 nm. Sample preparation and gradient elution were avoided by using a column‐switching technique. The sensitivity of the assay was excellent for clinical routine analysis, with a detection limit of 0.2 mg/L 5‐HIAA. No endogenous or exogenous interference problems arose. Intra‐ and interassay precision was good, with observed coefficients of variation of 1.5 to 2.6% and 2.1%, respectively. Recoveries were 93 to 98%. The system described can be used for clinical diagnosis and therapy follow‐up of carcinoid tumors. It has been running for over a year without disturbances and with a minimum of technical attendance.

Taga, Chiaki; Tsuji, Motohiro; Nakajima, Teruo

doi: 10.1002/bmc.1130030306pmid: 2765692

A reversed phase HPLC method with fluorometric detection for the analysis of β‐phenylethylamine has been developed using p‐methoxyphenylethylamine as an internal standard. Two columns, containing 200 μL of Dowex 50‐X8 and Amberlite CG‐50 respectively, were used to prepare a fraction containing β‐phenylethylamine. The recoveries of β‐phenylethylamine and p‐methoxyphenylethylamine were 53.9 ± 9.4% and 68.1 ± 12.4%, respectively, and elution profile of p‐methoxyphenylethylamine was sufficiently well correlated with that of β‐phenylethylamine. Regional distributions of β‐phenylethylamine in rat and mouse brains were determined. The highest concentrations were found in hypothalamus and hippocampus in both animals.

Bezwoda, W. R.; Mansoor, N.

doi: 10.1002/bmc.1130030307pmid: 2765693

The isolation and properties of lactoferrin from human breast milk and from neutrophilic granulocytes were investigated. Human breast milk lactoferrin was purified by means of heparin‐sepharose or Cibacron Blue affinity chromatography. Quantitative recovery using these two methods was comparable but Cibacron Blue affinity chromatography allowed for isolation of a more homogenous protein. Lactoferrin could only be isolated from human neutrophilic granulocytes by sequential use of antibody affinity followed by non‐specific affinity chromatography. Both breast milk lactoferrin and granulocyte lactoferrin were separated into apo and iron‐rich species by SDS polyacrylamide gel chromatography. Iron binding is accompanied by a conformational change in tertiary structure associated with more rapid electrophoretic migration. The isoelectric point of both human breast milk lactoferrin and human granulocyte lactoferrin is 5.5 – 6.2. Both types of lactoferrin have similar iron binding properties with release of iron from the one binding site occurring at pH 5.2 – 6.0 while the other binding site holds on to iron down to pH 3.6 – 3.2. Despite the high affinity for iron the percentage saturation of native lactoferrin is low, that for breast milk lactoferrin averaging 12 – 25% and that for granulocyte lactoferrin < 10%.

Korkolainen, Tapio; Nissinen, Erkki

doi: 10.1002/bmc.1130030308pmid: 2548643

The soluble form of catechol‐O‐methyltransferase (EC 2.1.1.6.) from rat liver was purified to homogeneity by high‐performance anion‐exchange chromatography and high‐performance gel‐filtration chromatography. The specific activity of the final pool was 270 U/mg protein. The purification was 1180‐fold and recovery of the enzyme activity was 15%. During this rapid and gentle purification there were no problems with loss of activity, and the estimated half life of the final purified enzyme pool was 5.5 days at +4°C. The only additive used was phenylmethylsulfonylfluoride in the homogenizing buffer.