Abstract: The sample solution (50 µL) was injected into a carrier solution (0.1 M pyrophosphate buffer of pH 8.7), then the flow was split between two reactors containing putrescine oxidase and xanthine oxidase, respectively, immobilized on controlled-pore glass. A delay coil was incorporated in the latter flow line so that two peaks would be obtained for each sample. The flows were reunited before introduction of a mediator solution [1.0 mM Fe(CN)64- in 0.1 M phosphate buffer of pH 7.0] and flow-through a mixing coil to a cell equipped with a vitreous carbon electrode on which peroxidase had been cross-linked by glutaraldehyde for amperometric measurement of the H2O2 formed in the two enzymatic reactions. The first and second signals corresponded to total polyamines (cadaverine, spermine and putrescine) and to hypoxanthine, respectively. The calibration graphs were rectilinear from 1 to 500 µM and the detection limit was 0.5 µM for polyamines or for hypoxanthine. Both reactors maintained adequate activity during repetitive use for 40 days. The method was applied to a deproteinized aqueous extract of pork loin meat undergoing storage at 5°C. A flow injection system is proposed for the determination of meat freshness, based on the simultaneous determination of polyamines and hypoxanthine. A putrescine oxidase reactor and xanthine oxidase reactor were incorporated at fixed positions in a flow system, which was based on the splitting of the flow after sample injection and subsequent confluence before reaching the peroxidase electrode. Because each channel has a different residence time, two peaks were obtained. The first peak corresponded to the total polyamine concentration. (putrescine, cadaverine and spermidine); the second peak to hypoxanthine. The calibration graphs were linear in the range 1 x 10^-6-5 x 10^-4M. The detection limit was 0.5 x 10^-6 M for polyamines and hypoxanthine. The measurement of polyamines and hypoxanthine contents in porcine loin meat could be performed at a rate of 25 samples per h with satisfactory precision (<1.5% RSD).

Abstract: Flow injection methods for sensitive, accurate and rapid determination of diamines, mainly putrescine (PUT) and cadaverine (CAD), and diamine oxidase (DAO) are described. Putrescine determination is based on an immobilized enzyme reactor (IER) loaded with putrescine oxidase and amperometric detection of the produced hydrogen peroxide at a platinum electrode. This assay was designed for the diagnosis of bacterial vaginosis. Putrescine oxidase (PUO) was assayed by simple injection of putrescine oxidase solution into a carrier buffer containing a fixed concentration. of putrescine followed by similar detection of hydrogen peroxide. In PUT measurements peak currents were linearly related to putrescine concentration. in the range from 10 µM to 1 mM with sample frequency of ~70 samples/h. No change in sensitivity and detection limit were observed after more than 1000 injections over a two week period. PUO was determined in the range of 50-2500 mU/mL, with sample frequency of ~40 samples/h. The lower detection limit of the present method (1.5 mU/mL) is suitable for determining the expected low levels of PUO in the amniotic fluid as a possible marker for premature rupture of membranes (PROM). The effects of flow rates, sample sizes, and substrate concentrations. in the detection limits and assay efficiencies are given in the present work. The efficacy of these assays for clinical diagnosis of the above-stated stated female reproductive tract disorders (bacterial vaginosis and amnion premature rupture) are discussed.

Abstract: A method is described for the HPLC analysis of the aliphatic polyamines agmatine, putrescine, cadaverine, spermidine and spermine in vegetables. Sample (1 g) was homogenised with 7 mL of 5% perchloric acid, cooled for 1 h at 0°C, and centrifuged. The supernatant was analyzed on a column (3.5 cm x 6 mm) of Polyaminepak (strong cation-exchange resin, 5 µm) with a mobile phase (0.65 mL min-1) of 1.0 M sodium citrate (pH 5.4) - acetonitrile (9:1). Post-column derivatization with phthalaldehyde (details given) and subsequent fluorescent detection was used.

Abstract: A convenient and highly sensitive analytical system was constructed for the estimation of microbial spoilage in meat. Putrescine and cadaverine produced by bacteria were degraded by an immobilized putrescine oxidase column and the hydrogen peroxide generated was determined by luminol chemiluminescence. Peroxidase from Arythromyces ramosus was used as a catalyst to obtain high sensitivity and a photodiode was used as the detector to construct a conventional chemiluminescence-flow injection analysis system. The calibration curves were linear in the 100 pmol/mL-200 nmol/L range for putrescine and 200 pmol/mL-80 nmol/mL for cadaverine. The coefficients of variation were 2.87%-3.15% for putrescine solution and 3.34% for the specimen solution. This system was able to detect putrescine and cadaverine after the bacterial count reached 4.3 P 10(6) cells/g in meat and the response increased with the increase in bacterial count. The regression equation between putrescine and cadaverine levels as determined by HPLC and this system was y=1.015x-0.501, and the correlation coefficient was 0.911.