Biomedical Chromatography

doi: 10.1002/bmc.1130080101pmid: N/A

Prados, Pablo; Higashidate, Sakae; Imai, Kazuhiro

doi: 10.1002/bmc.1130080102pmid: 8130626

A fully automated in‐line extraction reversed‐phase high‐performance liquid chromatography (HPLC) method with chemiluminescence detection was developed for the analysis of human and rat plasma catecholamines (CAs), norepinephrine (NE), epinephrine (E) and dopamine (DA). N‐Methyldopamine (N‐MeDA) was used as an internal standard. The method involves collection of plasma samples, which are first diluted with a sample dilution buffer containing N‐MeDA, and in‐line extraction of CAs using a carboxylic acid small resin precolumn (SERUMOUT‐CEX). This pre‐extraction process was coupled with an HPLC system including reversed‐phase mode separation on an analytical column (TSK gel ODS‐80Ts), fluorogenic derivatization with ethylenediamine (ED) and finally postcolumn peroxyoxalate chemiluminescence reaction detection using bis (4‐nitro‐2‐(3,6,9‐trioxadecyloxycarbonyl)phenyl)oxalate (TDPO) and hydrogen peroxide. The optimized mobile phase compositions, flow rates, operation timing for the adsorption and desorption of CAs in the precolumn, the separation in the analytical column and the optimum fluorogenic and chemiluminogenic reaction conditions were investigated. The detection limit for all the CAs was about 1 fmol (signal‐to‐noise ratio is 2). Excellent linearity of the calibration curves for CAs was observed in the range from 5 to 500 fmol for each CA using the internal standard. The relative standard deviations of the method for determining NE (183 fmol), E (23.6 fmol) and DA (6.1 fmol) in 50 μL of human plasma (n = 3) were 2.8, 2.7 and 3.1%, respectively, for the within‐day assay and 5.0, 3.8 and 4.0%, respectively, for the between‐day assay. The method was applicable to the determination of CAs in 25–50 μL of human or rat plasma.

Masuda, Kazuyoshi; Hirano, Koichiro; Takagishi, Yasushi

doi: 10.1002/bmc.1130080103pmid: 7510554

This paper describes a method suitable for purifying immunotoxin containing type 1 ribosome‐inactivating protein, gelonin. The separation of free (unreacted) 80G, a monoclonal antibody against α‐fetoprotein (AFP), from semipurified 80G–gelonin conjugate was unsuccessful by conventional CM‐Sepharose ion‐exchange chromatography because the isoelectric point of the conjugate did not increase enough to reach that of gelonin alone. In contrast, Blue Sepharose affinity chromatography could efficiently separate free 80G from the semipurified conjugate because the conjugate was bound to the column by its gelonin moiety while free 80G was not in buffer containing NaCl of a particular concentration range. However, a small amount of conjugate containing gelonin modified with N‐succinimidyl 3‐(2‐pyridyldithio)propionate, but not with 2‐iminothiolane, could not bind to the column. The conjugate purified by the use of Blue Sepharose showed selective cytotoxicity against AFP‐producing human hepatoma cells.

Kaku, Shinsuke; Tanaka, Makoto; Muramatsu, Makoto; Otomo, Susumu

doi: 10.1002/bmc.1130080104pmid: 7510553

A simple and sensitive assay method for NO synthase activity is described. Using glassy carbon as electrode and 30% methanol solution with 10 mM NH4Cl as mobile phase, NO 2− can be measured without disturbing ECD‐detectable substance in NO synthase assay mixture. The NO 2− production in the assay mixture of rat cerebellum NO synthase increased with protein and in a time‐dependent manner. The Km value for the substrate, L‐arginine, was 1.25 μM. The enzyme activity was inhibited in a concentration‐dependent manner by a NO synthase inhibitor, NNA. The Ki value for NNA was 0.166 ± 0.060 μM. This ECD–HPLC method for determining NO synthase activity has advantages compared with the diazo‐coupling method of the Greiss reagent and the isotopic method in which the conversion of the substrate, (14C)L‐arginine, to the product, (14C)L‐citrulline is measured; it is simple, sensitive and is convenient for studying the NO synthase activity with various compounds as the substrate.

Higashidate, Sakae; Imai, Kazuhiro; Prados, Pablo; Adachi‐Akahane, Satomi; Nagao, Taku

doi: 10.1002/bmc.1130080105pmid: 8130627

A calcium antagonist, diltiazem, was infused continuously into Sprague‐Dawley rats through the left femoral vein at four different flow rates. The mean arterial blood pressure and concentrations of plasma norepinephrine (NE) were measured in each single rat (n = 5) and the correlations between them were studied. Blood (150 μL) was collected 13 times during the infusion. Plasma NE was determined by HPLC–ethylenediamine condensation reaction–peroxyoxalate chemiluminescence detection system (HPLC–ED–PO‐CL). In four cases from 5 rats, the blood pressure reduction caused by diltiazem was inversely correlated to logarithm of plasma NE concentration. The relation was expressed as Y = −α logX + m. The coefficients of correlation were −0.9506, −0.9293, −0.9341 and −0.8675, respectively. The correlation for the last rat was worse (r = −0.0799). The good correlation would imply that the sympathetic nervous system released NE to maintain blood pressure up to the normal level, responding to the blood pressure reduction caused by diltiazem. The present experiment proved the feasibility of the determination method of NE utilizing HPLC–ED–PO‐CL detection in applying to the individual rats.

Aboul‐Enein, Hassan Y.; Serignese, Vince

doi: 10.1002/bmc.1130080106pmid: 8130628

A direct, isocratic and simple chromatographic method is described for the enantiomeric separation of bevantolol (BEV) using normal and reverse cellulose tris (3,5‐dimethylphenylcarbamate) chiral stationary phases (CSPs) known as Chiralcel OD and Chiralcel OD‐R, respectively. The effect of various alcohols present in the mobile phase on the separation factor (α) and resolution factor (Rs) has been studied. The method has been used to determine and identify the BEV enantiomers in human urine after oral administration of racemic BEV. The chiral recognition mechanism(s) between the analyte and these chiral stationary phases is proposed.

Andeejani, Ahmed M. I.; Hughes, Herbert; Feuchuk, Danny M.; Aboul‐Enein, Hassan Y.

doi: 10.1002/bmc.1130080107pmid: 8130629

A rapid method for the determination of iopanoic acid (IOP) in dog plasma utilizing a Hisep column was developed. A mobile phase of 12% methanol, 88% 0.05 M phosphate buffer pH 3.4 yielded a k′ of 8.5 with no interference from proteins present in plasma. Recoveries of IOP from spiked plasma ranged from 97% to 103% at 270 μmol/L and 1.75 mmol/L respectively. Replication was ±2.8% at 1.75 mmol/L and ±6% at 21 μmol/L. A method utilizing 2,4,6‐triiodobenzoic acid as internal standard was also developed for comparison.

Delbeke, F. T.; Debackere, M.

doi: 10.1002/bmc.1130080108pmid: 8130630

A high performance liquid chromatographic method to measure plasma and urine fenoprofen levels in equine biofluids is described. Liquid–liquid extraction with diethylether was used to isolate the drug from plasma and urine. The accuracy and reproducibility of the method were within acceptable limits over the concentration range 0–10 μg/mL and 0–20 μg/mL respectively from plasma and urine. Detection limits were 0.05 μg/mL (2 mL plasma) and 0.2 μg/mL (0.5 mL urine). This procedure was applied to ascertain the pharmacokinetics of a 3 g dose of fenoprofen calcium in a horse.

Dimov, N.; Simeonov, S.

doi: 10.1002/bmc.1130080109pmid: 8130631

Experimentally defined relationships have been found to describe adequately the retention and separation of bovine and porcine insulins as well as their desamido products on reversed phases C4 30 nm and C18 10 nm pore diameter. The equations are valid for a region of initial acetonitrile concentrations from 16 to 31% and gradient rates from 0.04 to 0.60%/min. The peak heights showed an exact non‐linear relationship with the time interval between the first and last peak of interest, independent of their retention time. The number of experiments required for obtaining the parametric estimates of the models depends on the particular task, but in all cases is less than eight. The relationships found permit the correct choice to be made in advance both for analysis conditions for a particular column and for different requirements of insulin analysis.

Catalin, Jacques; Peloux, Anne F.; Coloma, Fabrice; Payet, Brigitte; Lacarelle, Bruno; Cano, Jean P.; Just‐Landi, Sylvaine

doi: 10.1002/bmc.1130080110pmid: 8130632

A high‐performance liquid chromatography method which uses direct injection and a column‐switching valve for determination of mitoxantrone in plasma is described. After addition of internal standard, plasma was deproteinized by adding 5‐sulphosalicylic acid reagent. The supernatant was injected onto an enrichment precolumn flushed with washing solvent (methanol and water 5:95). Absorbed mitoxantrone was backflushed from the precolumn into an analytical column C18 Nucleosil 250 × 4 mm with a gradient elution (solvent A, ammonium formate buffer 1.6 M, pH 4.3; solvent B, acetonitrile and water 40:60; linear gradient from 45 to 55% of B for 30 min was programmed) at a flow rate of 1.3 mL/min. Detection was carried out at 665 nm. This method showed obvious advantages over conventional extraction procedures in terms of speed and facility of sample handling.