Rapid detection of the active cardiac glycoside convallatoxin of lily of the valley using LOCI digoxin assay.

OBJECTIVES: To explore the luminescent oxygen channeling technology-based digoxin immunoassay (LOCI digoxin assay) for rapid detection of lily of the valley extract and convallatoxin. The potential in vitro binding of convallatoxin with Digibind was also evaluated. METHODS: Aliquots of a drug-free serum pool and a digoxin serum pool were supplemented with lily of the valley extract or convallatoxin, and then apparent digoxin concentrations were measured using the LOCI digoxin assay. Mice were administered lily of the valley extract or 50 µg of convallatoxin, and digoxin concentrations in serum specimens were measured 1 and 2 hours after gavage. Aliquots of a serum pool supplemented with convallatoxin or lily of the valley extract were further supplemented with various concentrations of Digibind and free apparent digoxin concentrations were measured. RESULTS: Apparent digoxin concentrations were observed when aliquots of a drug-free serum pool were supplemented with convallatoxin or lily of the valley extract, and also with convallatoxin or herbal extract. Bidirectional interference of convallatoxin and lily of the valley extract with serum digoxin measurement using the LOCI assay was also observed. Digibind was capable of binding convallatoxin in vitro. CONCLUSIONS: LOCI digoxin assay can be used for rapid detection of convallatoxin, and Digibind can bind convallatoxin in vitro.

Rapid detection of convallatoxin using five digoxin immunoassays.

CONTEXT: Cardiac glycosides of plant origin are implicated in toxic ingestions that may result in hospitalization and are potentially lethal. The utility of commonly available digoxin serum assays for detecting foxglove and oleander ingestion has been demonstrated, but no studies have evaluated the structurally similar convallatoxin found in Convallaria majalis (lily of the valley) for rapid laboratory screening, nor has digoxin immune Fab been tested as an antidote for this ingestion. OBJECTIVE: We aimed to (1) evaluate multiple digoxin assays for cross-reactivity to convallatoxin, (2) identify whether convallatoxin could be detected in vivo at clinically significant doses, and (3) determine whether digoxin immune Fab could be an effective antidote to convallatoxin. MATERIALS AND METHODS: Cross-reactivities of purified convallatoxin and oleandrin with five common digoxin immunoassays were determined. Serum from mice challenged with convallatoxin was tested for apparent digoxin levels. Binding of convallatoxin to digoxin immune Fab was determined in vitro. RESULTS: Both convallatoxin and oleandrin were detectable by a panel of commonly used digoxin immunoassays, but cross-reactivity was variable between individual assays. We observed measurable apparent digoxin levels in serum of convallatoxin intoxicated mice at sublethal doses. Convallatoxin demonstrated no binding by digoxin immune Fab. CONCLUSION: Multiple digoxin immunoassays detect botanical cardiac glycosides including convallatoxin and thus may be useful for rapid determination of severe exposures, but neutralization of convallatoxin by digoxin immune Fab is unlikely to provide therapeutic benefit.

LC-ESI-MS/MS characterization of strophanthin-K.

A liquid chromatography-mass spectrometry (LC-MS) method was developed for the characterization of strophanthin-K, a mixture of cardiac glycosides extracted from the seeds of Strophanthus kombe. The method is based on the separation of the cardenolides using high performance liquid chromatography (HPLC) followed by detection with electrospray ionization mass-spectrometry (ESI-MS). Chromatographic separation of the analytes was achieved on a RP C-18 column using water: 1% formic acid in water (v/v):acetonitrile gradient mobile phase. Strophanthin-K glycosides studied in ESI-MS in negative ion mode formed abundant adduct ions [M+HCOO](-) while the pseudomolecular ions [M-H](-) are obtained in ESI-MS/MS experiments. Six different cardiac glycosides were identified and characterized: k-strophanthoside, k-strophanthin-beta, helveticoside (erysimin), erysimoside, cymarin and neoglucoerysimoside. Forced degradation investigations done with strophanthin-K showed that k-strophanthidin (the aglycone of strophanthin-K glycosides) was the main product of degradation in acidic conditions; however, in basic conditions, the hydrolysis of the unsaturated 17beta-lactones to the corresponding gamma-hydroxy acids was the predominant degradation pathway.

[False positive and false negative results in testing for pyrogenic impurities]. / Falschpositive und falschnegative Resultate bei der Prüfung auf pyrogene Verunreinigungen.

Causes of the occurrence of pyrogenic impurities in injection and infusion preparations as well as false-positive and false-negative results possible in the pyrogen test are reported. Possibilities of excluding pyrogenic impurities in the preparations concerned and of avoiding false-positive and false-negative results in the pyrogen test are discussed.

[Optimization of the use of horseradish peroxidase and its antibodies in immunoenzyme analysis]. / Optimizatsiia ispol'zovaniia peroksidazy khrena i ee antitel v immunofermentnom analize.

The steady-state kinetics of peroxidation of 8 aromatic amines was studied. p-Phenylenediamine, o-dianisidine (o-DA) and 3,5,3',5'-tetramethylbenzidine were found to be optimal substrates of horse-radish peroxidase. The kinetics of oxidation of these substrates by horseradish peroxidase modified with three molecules of Strophanthin K was studied as well. Within the temperature range from 37 to 53 degrees C the inactivation rate constants were determined for peroxidase and its conjugate with Strophanthin K. The effect of sugars and polyols on thermal stability of the conjugate peroxidase-Strophanthin K was investigated. A comparative kinetic study was performed of oxidation of o-DA and its conjugate with dextran. The results obtained made a basis for an enzyme immunoassay of cardiac glycosides during their isolation from plant raw material.

[K-strophanthin-beta complexing with calcium, magnesium and dysprosium ions]. / Kompleksoobrazovanie k-strofantina-beta s ionami kal'tsiia, magniia i disproziia.

Nuclear magnetic resonance, microcalorimetry and the use of the ion-selecting electrode evidenced that k-strophanthine-beta forms complexes with the calcium, magnesium and disprosium ions. Changes in the position of the k-strophanthine-beta carbohydrate parts and aglycones signals bear witness to their participation in the complexing.