Einar Jensen

Quantitative analysis of flavonoids by reversed-phase high-performance liquid chromatography

Abstract Large variations were found in the effectiveness of five reversed-phased high-performance liquid chromatography columns used for the analysis of free and conjugated flavonoids. The best results were obtained with C 18 Nova-Pak, C 18 Symmetry and C 18 Genesis columns but substantial band broadening and peak asymmetry were observed when free flavonoids were chromatographed on ODS-Hypersil and LiChrospher RP-18 supports. The Symmetry and Genesis columns provided gradient elution separations of rutin, quercetin-3-glucoside, quercitrin, myricetin, luteolin, quercetin, apigenin, kaempferol and isorhamnetin. This procedure was used for the quantitative analysis of endogenous flavones and flavonols in acid hydrolysed extracts from onions and celery.

Occurrence of selective serotonin reuptake inhibitors in sewage and receiving waters at Spitsbergen and in Norway

A method for the determination of five selective serotonin reuptake inhibitors (citalopram, sertraline, fluoxetine, fluvoxamine and paroxetine) and four of their metabolites (desmethylcitalopram, didesmethylcitalopram, norfluoxetine and desmethylsertraline) in seawater and sewage influents and effluents, has been developed and validated. The method is based on a three-phase hollow-fibre supported liquid phase microextraction of 1.1L samples, followed by high performance liquid chromatography with electrospray ionization and mass spectrometric detection. The detection limits varied between 17 pg/L (citalopram) and 618 ng/L (desmethylsertraline), and the quantification limits between 57 pg/L (citalopram) and 4.1 ng/L (desmethylsertraline). Sampling was done from February to August in 2007 on three different locations with dissimilarities concerning waste water treatment procedures. No significant difference in SSRI cleansing efficiency between merely sieving (Langnes STP, Tromsø) and a more advanced sewage treatment (VEAS STP, Oslo) was seen. All the investigated compounds are present in all waste water samples from these STPs, and a total concentration of SSRIs and metabolites up to 840 ng/L has been found. Untreated sewage samples have been collected in the small town Longyearbyen at Spitsbergen. Despite few inhabitants (2000), it was still possible to find traces of SSRIs in the waste water. In Tromsø and Longyearbyen the waste water is discharged into the sea, therefore seawater samples have been collected close to the outlets. The results show higher concentrations of SSRIs outside Longyearbyen than Tromsø, possibly due to the stronger tidal currents around Tromsø. However, the concentrations are quite low, not exceeding total concentrations of 3 ng/L.

Different digestion of caprine whey proteins by human and porcine gastrointestinal enzymes

The objective of the present study was twofold: first to compare the degradation patterns of caprine whey proteins digested with either human digestive juices (gastric or duodenal) or commercial porcine enzymes (pepsin or pancreatic enzymes) and second to observe the effect of gastric pH on digestion. An in vitro two-step assay was performed at 37 degrees C to simulate digestion in the stomach (pH 2, 4 or 6) and the duodenum (pH 8). The whey proteins were degraded more efficiently by porcine pepsin than by human gastric juice at all pH values. Irrespective of the enzyme source, gastric digestion at pH 2 followed by duodenal digestion resulted in the most efficient degradation. Lactoferrin, serum albumin and the Ig heavy chains were highly degraded with less than 6 % remaining after digestion. About 15, 56 and 50 % Ig light chains, beta-lactoglobulin (beta-LG) and alpha-lactalbumin remained intact, respectively, when digested with porcine enzymes compared with 25, 74 and 81 % with human digestive juices. For comparison, purified bovine beta-LG was digested and the peptide profiles obtained were compared with those of the caprine beta-LG in the digested whey. The bovine beta-LG seemed to be more extensively cleaved than the caprine beta-LG in the whey. Commercial enzymes appear to digest whey proteins more efficiently compared with human digestive juices when used at similar enzyme activities. This could lead to conflicting results when comparing human in vivo protein digestion with digestion using purified enzymes of non-human species. Consequently the use of human digestive juices might be preferred.

Analysis of 3-indole carboxylic acid in Pinus sylvestris needles

Abstract 3-Indole carboxylic acid (ICA) has been characterized as an endogenous constituent of Pinus sylvestris needles. Quantitative estimates of 3-indole acetic acid (IAA) and ICA, corrected for both sample losses and the conversion of IAA to ICA occurring during purification, indicate that Pinus needles contain 24.5 ± 6.5 ng IAA/g and 2.3 ± 0.4 ng ICA/g.

Cucurbic acid and its 6,7-stereoisomers

Abstract The derivatives 7- iso -cucurbic acid (7- iso -CA) and 6- epi -7- iso -cucurbic acid (6- epi -7- iso -CA) were prepared from (−)-jasmonic acid (JA) by reduction and cucurbic acid (CA) and 6- epi - cucurbic acid (6- epi -CA) from (+)-7- iso -jasmonic acid (7- iso -JA). The chromatographic properties (TLC, HPLC, GC) of these derivatives are described and the structures established by physical data. The extracts of different plant materials were analysed with respect to the natural occurrence of JA and CA and its 6,7-stereoisomers. JA, 6- epi -CA and 6- epi -7- iso -CA were identified in female flowers of Juglans regia and JA, CA, 6- epi - CA and 6- epi -7- iso -CA in spores of Anemia phyllitidis . JA, CA, 6- epi -CA and 9, 10-dihydro-JA were detected in immature caryopses of Secale cereale . In rye JA dominated in young caryopses, while CA level exceeded the amount of JA in the premature fruits. In each tissue 6- epi -CA was found to be a minor component, while CA or 6- epi -7- iso - CA represented the major components; 7- iso -CA could not be detected.

Depletion of selective serotonin reuptake inhibitors during sewage sludge composting.

Sewage and sewage sludge is known to contain pharmaceuticals, and since sewage sludge is often used as fertilizer within agriculture, the reduction of the selective serotonin reuptake inhibitors (SSRIs) Citalopram, Sertraline, Paroxetine, Fluvoxamine and Fluoxetine during composting has been investigated. Sewage sludge was spiked with the SSRIs before the composting experiment started, and the concentration of the SSRIs in the sludge during a 21 day composting period was measured by liquid phase microextraction (LPME) and high-performance liquid chromatography-mass spectrometry. All the SSRIs had a significant decrease in concentration during the composting process. The highest reduction rates were measured for Fluoxetine and Paroxetine and the lowest for Citalopram. In addition three out of four known SSRI metabolites were found in all the samples, and two of them showed a significant increase in concentration during the composting period.

Amino acid conjugates as metabolites of the plant growth regulator dihydrojasmonic acid in barley (Hordeum vulgare)

The biotransformation of [2-14C](±)9, 10-dihydrojasmonic acid (DJA) was studied in excised shoots of 6-day-old barley seedlings after 72 h. From the ethyl acetate extract, some minor metabolites were isolated and purified by DEAE-Sephadex A-25 chromatography, thin-layer chromatography (TLC), C18-cartridges, and high-performance liquid chromatography (HPLC). The structural identification of these metabolites was performed by gas chromatography-mass spectrometry (GC-MS), circular dichroism (CD), and amino acid analysis, and the following amino acid conjugates were found:N-[(−)9,10-dihydrojasmonoyl]valine,N-[(−)9,10-dihydrojasmonoyl]isoleucine,N-[9,10-dihydrojasmonoyl]leucine,N-[11-hydroxy-9,10-dihydrojasmonoyl]valine,N-[11-hydroxy-9,10-dihydrojasmonoyl]isoleucine,N-[12-hydroxy-9,10-dihydrojasmonoyl]isoleucine; and the cucurbic acid-related compoundsN-{[3-hydroxy-2(4-hydroxypentyl)-cyclopent-1-yl]-acetyl}isoleucine andN-{[3-hydroxy-2(5-hydroxypentyl)-cyclopent-1-yl]-acetyl}isoleucine. The results suggest conjugation with isoleucine and valine, as well as preferential hydroxylation at position C-11 or hydrogenation at position C-6, as being important steps in the metabolism of (±)DJA in barley shoots.

Analysis of gibberellins and gibberellin conjugates by ion-suppression reversed-phase high-performance liquid chromatography

Abstract Ion-suppression reversed-phase high-performance liquid chromatography of 42 gibberellins and 20 gibberellin glucosides and glucosyl esters has been investigated using a C 18 support eluted isocratically with a range of methanol concentrations in aqueous phosphoric acid at pH 3.0. Detection was with an absorbance monitor operating at 208 nm. The data obtained enables correlations to be made between chromatographic retention properties and gibberellin structure.

Biosynthesis of indole-3-acetic acid in protoplasts, chloroplasts and a cytoplasmic fraction from barley (Hordeum vulgare L.)

Protoplast preparations from barley (Hordeum vulgare L.) enzymatically converted [5-3H]tryptophan to [3H]indole-3-acetic acid (IAA). Both a chloroplast and a crude cytoplasmic fraction, isolated from protoplasts that had previously been fed [5-3H]tryptophan, contained [3H]IAA. Chloroplast and cytoplasmic preparations, isolated from protoplasts and thereafter incubated with [5-3H]tryptophan, also synthesized [3H]IAA, although, in both instances the pool size was less than 50% of that detected in the in-vivo feeds. There were no significant differences in the amounts of [3H]IAA that accumulated in protoplast and chloroplast preparations incubated in light and darkness.

High-performance liquid chromatographic separations of naphthoquinones and their derivatives: Effect of hydrogen bonding on retention

A method for the separation and determination of fifteen naphthoquinone derivatives was developed, based on reversed-phase high-performance liquid chromatography and ultraviolet-visible detection. The effect on the selectivity of different mobile phase compositions (e.g., methanol-water and acetonitrile-water binary mixtures and methanol-acetonitrile-water ternary mixture) was investigated. The retention order of the compounds with methanol-water as eluent is interpreted on the basis of intramolecular hydrogen bonding in the solute versus intermolecular hydrogen bonding between the solute and the solvent.The hydrogen bonding pattern was studied using quantum chemical calculations.