Rapid, presumptive identification of seed-based toxins using direct analysis in real time mass spectrometry (DART-MS) and its variants.

Detection of seed-based toxins is a need for forensic chemists when suspected poisonings occur. The evidence that is found is often physically unidentifiable, as the seeds are mashed to extract the toxin. This work investigates potential strategies for rapid detection of seed-based toxins and seed mashes containing these toxins using chemical signatures obtained by direct analysis in real time mass spectrometry (DART-MS). Seven toxins (digoxin, digitoxin, hypaconitine, hyoscyamine, lanatoside, oleandrin, and scopolamine) and six seeds containing these toxins were studied. While detection of four of the toxins was readily attainable, detection of digoxin, digitoxin, and lanatoside was hindered by the inability to thermally desorb these larger compounds under normal operating conditions. The use of DART-MS variants capable of higher desorption temperatures (thermal desorption (TD)-DART-MS and infrared thermal desorption (IRTD)-DART-MS) enabled detection of these compounds. Detection of toxins from direct analysis of seed mashes and methanolic seed mash extracts was found to be compound and technique dependent. Principal component analysis (PCA) of generated mass spectra enabled differentiation of seed species, even in cases where the toxins were undetectable.

Rapid quantification of digitoxin and its metabolites using differential ion mobility spectrometry-tandem mass spectrometry.

This study focuses on the quantitative analysis of the cardiac glycoside drug digitoxin and its three main metabolites digitoxigenin-bisdigitoxose, digitoxigenin-monodigitoxose, and digitoxigenin using electrospray ionization-differential ion mobility spectrometry-tandem mass spectrometry (ESI-DMS-MS/MS). Despite large molecular weight differences, gas-phase separation of the four compounds in the DMS drift cell was not possible, even by utilizing additional volatile chemical modifiers. Baseline separation was achieved after adduct formation with alkali metal ions, however, and efficiency was shown to improve with increasing size of the alkali ion, reaching optimum conditions for the largest cesium ion. Subsequently, an assay was developed for quantification of digitoxin and its metabolites from human serum samples and its analytical performance assessed in a series of proof-of-concept experiments. The method was applied to spiked human serum pools with concentration levels between 2 and 80 ng/mL. After a short reversed-phase chromatographic step for desalting the sample, rapid DMS separation of the analytes was carried out, resulting in a total run time of less than 1.5 min. The instrumental method showed good repeatability; the calculated coefficients of variation ranged from 2% to 13%.

HPLC method validation for Digitalis and its analogue by pulsed amperometric detection.

We developed a highly sensitive and selective reversed-phase HPLC-pulsed amperometric detection (RP-HPLC-PAD) method for cardiac glycoside detection. Eight cardiac glycosides were completely separated within 45 min on a reversed-phase column using a water-acetonitrile gradient, and were detected using a PAD under NaOH alkaline conditions. The detection (S/N=3) and quantification (S/N=10) limits for the cardiac glycosides were 0.1-0.3 and 0.3-0.8 ng, respectively. The linear regression coefficient was 0.9962-0.9998 for concentrations of 1-25 µg/mL. Cardiac glycosides in the Digitalis purpurea leaf displayed intra- and inter-day precisions (RSDs) of <9.30% and average recoveries of 98.63-99.94%. The contents of gitoxin, digitonin, and digitoxin in the D. purpurea were 0.197, 0.11, and 0.379 mg/g for leaf dried at 60 °C, 0.058, 0.11, and 0.090 mg/g for leaf dried at ambient temperature, and N.D. (not detected), and 18.379 mg/g, N.D. for seed, respectively. We conclude that our method shows good precision and accuracy.

A novel LC-IDMS/MS method for the determination of the cardiac glycosides digoxin and digitoxin using caesium adducts.

This article describes an essential improvement of the published candidate reference measurement procedure for digoxin and digitoxin and compares it with the original method. The novelty of the method lies in the measurement of the caesium (Cs+) ion as product ion in the multiple reaction monitoring mode (MRM) with potentially improved analytical specificity whilst retaining a comparable accuracy and precision at therapeutic levels. The original measurement procedure used the single-ion mode (SIM). The dissociation of the Cs+ adducts in MRM leads to the formation of Cs+ ions as main charged product in high yield. The present method results in a product ion signal intensity in MRM for digoxin and digitoxin of up to 80% of the precursor ion signal intensity in SIM. The precision, expressed as the coefficient of variation of the new method for digoxin was 3.18% (SIM) and 2.28% (MRM) at a concentration of 0.66 microg/l and 1.26% (SIM) or 1.65% (MRM) at 2.0 microg/l. The corresponding data for digitoxin were 1.21% (SIM) and 1.62% (MRM) at 24 microg/l and 1.46% (SIM) and 1.13% (MRM) at 42 microg/l.

The Fab fragment of anti-digoxin antibody (digibind) binds digitoxin-like immunoreactive components of Chinese medicine Chan Su: monitoring the effect by measuring free digitoxin.

Chan Su, a Chinese medicine prepared from the skin glands of Chinese toads, is used in the treatment of cardiovascular diseases. Severe toxicity and even death has been reported from overdose with Chan Su. The cardiotonic effect of Chan Su is attributed to bufadienolides, which also have apparent digitoxin activity. We demonstrated that these components of Chan Su could be neutralized by digibind, both in vitro and in vivo. For in vitro experiments, we supplemented drug-free serum pools with aqueous extract of Chan Su. Then, to aliquots of serum pool containing Chan Su, various amounts of digibind (10, 25 or 50 microg/ml of serum) were added. After incubation, total and free digitoxin concentrations (in the protein-free ultrafiltrate) were measured using the fluorescence polarization immunoassay (FPIA) and a FLX/TDx analyzer. For in vivo experiments, mice were fed with Chan Su by gavage. After 45 min, 200 microg of digibind was administered by injection. Fifteen minutes after injection, blood was collected for analysis of total and free apparent digitoxin activities. We observed complete removal of apparent digitoxin activity from protein-free ultrafiltrate both in vitro and in vivo by digibind, indicating that digibind successfully binds Chan Su. We conclude that digibind neutralizes Chan Su, and measuring the free digitoxin concentrations can monitor such an effect.

Interference from digitoxin-like immunoreactive factors reduced in a new monoclonal chemiluminescent digitoxin assay.

Endogenous digoxin-like immunoreactive factors (DLIF) can interfere with some digoxin immunoassays. We looked for similar interference, called digitoxin-like immunoreactive factors (DTLIF) in two digitoxin immunoassays: A new chemiluminescent assay (CLIA), processed on the automated random access immunoassay system ACS:180, and a fluorescent polarization assay (FPIA), processed on the semiautomated TDx batch analyzer. One hundred thirty-seven samples of sera were tested from nondigitalized pregnant women, patients with liver or kidney diseases, and cord blood. The CLIA digitoxin assay uses a murine monoclonal antibody and requires no sample pretreatment; the FPIA digitoxin assay uses a polyclonal rabbit antibody and requires sample precipitation. Both assays have a similar dynamic range and sensitivity and give comparable results with commercial controls and external quality control survey samples. Although the CLIA detected no digitoxin in any sample tested, the FPIA showed apparent digitoxin concentrations of more than 2.0 ng/ml for 100% and 44% among cord blood and liver disease specimens, respectively. The highest DTLIF concentration was found in serum from a patient with liver disease (18.1 ng/ml). When spiked with 32 ng/ml digitoxin, six of the samples containing DTLIF generated FPIA digitoxin values of 6% to 27.5% more than the expected digitoxin levels. Two specimens with no detectable DTLIF activity were run as controls, and when spiked with digitoxin, showed target digitoxin concentrations in the FPIA. The CLIA recovered near the target digitoxin values (32 ng/ml) in all spiked samples. It was concluded that the polyclonal FPIA digitoxin assay may give discordant digitoxin concentrations in some patient groups because of interference from digitoxin-like immunoreactive factors. The CLIA digitoxin assay is not affected by DTLIF interference.

Neutralization of cardiac toxins oleandrin, oleandrigenin, bufalin, and cinobufotalin by digibind: monitoring the effect by measuring free digitoxin concentrations.

Oleandrin plant poisoning is common in children and the plant extract is used in Chinese medicines. The toxicity is due to oleandrin and the deglycosylated metabolite oleandrigenin. Bufalin and cinobufotalin (toad cardiac toxins) are also widely used in Chinese medicines like Chan SU, and Lu-Shen -WU. Severe toxicity from bufalin after consumption of toad soup has been reported. Taking advantage of structural similarities of these toxins with digitoxin, we demonstrated that these compounds can be rapidly detected in blood by the fluorescence polarization immunoassay for digitoxin. The cross reactivities of these compounds with digoxin assay were much lower. For example, when a drug free serum was supplemented with 10 microg/ml of oleandrin, we observed 127.7 ng/ml of digitoxin equivalent but only 2.4 ng/ml of digoxin equivalent concentration. Digibind neutralized all cardiac toxins studied as evidenced by significant fall of free concentrations. When aliquots of serum pool containing 50.0 microg/ml of oleandrin were supplemented with 0, 10.0, 25.0, 50.0, 100, and 200 microg/ml of digibind, the mean free concentrations were 30.6, 23.3, 16.0, 10.7, 7.8 and 5.5 microg/ml respectively. Similarly, with 50.0 microg/ml of oleandrigenin (total concentration: 36.2 ng/ml), the free concentration was 14.5 ng/ml digitoxin equivalent in the absence of digibind and 5.4 ng/ml in the presence of 200 microg/ml of digibind. In another specimen containing 500 ng/ml bufalin (total concentration: 156.9 ng/ml), the free concentration was 8.6 ng/ml in the absence of digibind and none detected in the presence of 100.0 microg/ml digibind. Because such neutralization may also occur in vivo, digibind may be useful in treating patients exposed to these toxins.

Ultra-specific immunoassays for small molecules: roles of wash steps and multiple binding formats.

New immunometric forms of immunoassay are much more flexible to use than competitive-format immunoassays for small molecular analytes. An example of the utility of this flexibility is the ability to wash the capture antibody after it has been exposed to analyte but before addition of the labeled reagent. This simple maneuver has a large impact on the specificity obtained from already highly specific assays. We also show that specificity can be further increased by means of our multiple binding assay approach, in which the final reading reflects analyte binding to two different primary capture monoclonal antibodies.

[Digitoxin Reference Standard (Control 951) of the National Institute of Health Sciences].

The raw material of digitoxin was tested for preparation of the "Digitoxin Reference Standard (Control 951)". Analytical data obtained were as follows: loss on drying, 0.0%; infrared spectrum, the same as that of the JP Digitoxin Reference Standard (Control 845); thin-layer chromatography, no impurity was detected; high-performance liquid chromatography (HPLC), two kinds of impurities were detected and the total amount was estimated to be 0.16 +/- 0.01% (n = 3); assay, 99.0% by HPLC. Based on the above results, the raw material was authorized as the JP Digitoxin Reference Standard (Control 951).

Determination of cardiac glycosides in digitoxin tablets and deslanoside injections by micro-HPLC.

A micro high-performance liquid chromatographic (micro-HPLC) procedure for the assay of digitoxin tablets and deslanoside injections has been developed. Micro-HPLC is performed on an ODS micro column, with acetonitrile-methanol-water (10:20:17) for digitoxin tablets and acetonitrile-water (21:70) for deslanoside injections. The effluent is monitored by UV absorption at 220 nm. Quantitation of cardiac glycosides in tablets and injections is carried out by the internal standard method. The composite assay results for digitoxin tablets and deslanoside injections provide average values of 101.2 and 99.9% with standard deviations of 1.2 and 0.93%, respectively. This micro-HPLC method is sensitive, quantitative, and reproducible. It is suitable for use in examining the content uniformity of pharmaceutical preparations.

Identificaçäo cromatográfica de glicosídeos cardiotônicos em comprimidos e injetáveis / Chromatographic identifications of cardiac glycosides in tablets and injections

Os princípios ativos de comprimidos e injetáveis contendo glicosídeos cardiotônicos (digitoxina, digoxina e lanatosídeo C) foram identificados por Cromatografia em Camada Delgada

Lack of endogenous crossreactivity with three digitoxin radioimmunoassays in adults with renal insufficiency.

The possibility of interference by apparent digitoxin-like immunoreactive substance (DTLIS) with three radioimmunoassays was studied in patients with renal insufficiency. From each of 25 adult patients with renal insufficiency and 25 age-matched and sex-matched control subjects with normal renal function, a single serum sample was obtained and assayed for digitoxin content by three commercially available radioimmunoassays (GammaCoat, Coat-A-Count, and the Wien assay). Although two of the three assays found measurable concentrations, the difference in apparent digitoxin concentrations between the control subjects and those with renal insufficiency was not significant. Assay interference could not be explained on the basis of differences in age, serum creatinine concentration, or weight. The magnitude of DTLIS interference in relation to the digitoxin therapeutic range appears to be small with the radioimmunoassays used in this study.

Laser desorption/fourier transform ion cyclotron resonance mass spectrometry: digoxin, digitoxin, and their reduced and sugar-hydrolyzed metabolites.

Mass spectra of digoxin and digitoxin (the most widely prescribed drugs for treatment of congestive heart failure) and a complete set of their 14 dihydro- and sugar-hydrolyzed metabolites have been obtained via laser desorption/ionization with a Fourier transform ion cyclotron resonance (LD/FT/ICR) mass spectrometer. The most intense peak is typically the pseudomolecular [M + K]+ ion, but fragment ions corresponding to loss of 1-3 sugars and hydroxyls are also observed. LD/FT/ICR mass spectra for all 16 compounds were produced with a single set of sample and spectrometer parameters. No matrix peaks are present. Finally, LD/FT/ICR provides dynamic mass accuracy within approximately 5 ppm throughout a mass range of 404 less than m/z less than 819.

Comparison of digitoxin and its major glucuronidated metabolite: inhibition of Na-K-ATPase and reactivity with the radioimmunoassay.

The metabolism of digitoxin is known to occur through several major pathways including oxidation and glucuronidation. Several studies have compared the behavior of the various unconjugated metabolites with respect to reactivity toward Na-K-ATPase and toward the radioimmunoassay (RIA). Other studies with conjugated products synthesized chemically have also been performed. However, the activity of the conjugated products produced in vivo has not been reported and these metabolites are generally assumed to be inactive. In the present studies we have prepared and purified the glucuronide conjugate of digitoxigenin monodigitoxoside formed by rat liver microsomes and determined the activity of this metabolite as measured by inhibition of Na-K-ATPase and by the RIA. The concentration of the conjugated product needed to cause a fifty per cent inhibition of Na-K-ATPase was 5.40 microM compared to 0.68 for digitoxin and 0.08 for digitoxigenin monodigitoxoside. However, the conjugated product had a two-fold greater affinity for the antibody in the RIA procedure than did either digitoxin or digitoxigenin monodigitoxoside. Although these data cannot be extrapolated to the effects of these drugs on cardiac contractility, the results suggest that the contribution of the glucuronide conjugate to the therapeutic or toxic effect of digitoxin may be minimal. This metabolite may, however, lead to inaccurate estimation of blood levels by the RIA.

Detection of digoxin, digitoxin, their cardioactive metabolites and derivatives by high-performance liquid chromatography and high-performance liquid chromatography-radioimmunoassay.

High-performance liquid chromatographic (HPLC) systems are described for the separation of the cardioactive metabolites of the digoxin and digitoxin series that are formed by the splitting of digitoxose sugar residues from the aglycone steroid: isocratic separation of digoxin and its cardioactive metabolites; isocratic separation of digitoxin and its cardioactive metabolites; and gradient elution separation of the digoxin and digitoxin series including beta-acetyl- and beta-methyldigoxin. Separations were performed on a 10-microns bonded octadecyl phase column using various mixtures of acetonitrile--water and acetonitrile--methanol--water as the mobile phase. These methods provide high peak resolution and are well suited for collecting elution fractions, e.g. to link up with sensitive immunological measurements. An HPLC-radioimmunoassay method is described for the quantitation of digoxin, digitoxin and their metabolites in human tissues.

Chemiluminescent enzyme immunoassay using beta-D-galactosidase as the label and the bis(2,4,6-trichlorophenyl)oxalate-fluorescent dye system.

A chemiluminescent enzyme immunoassay (EIA) using beta-D-galactosidase as the enzyme label is described in this report. The activity of beta-D-galactosidase, after separation of bound and free fractions, was measured using a coupled enzyme procedure: lactose and glucose oxidase were used as the substrate and coupling enzyme, respectively. Hydrogen peroxide generated from glucose was determined by the chemiluminescence reaction using the bis(2,4,6-trichlorophenyl)oxalate-fluorescent dye system. This assay system was applied to the EIA of phenytoin using beta-D-galactosidase as the label and assay sensitivity was increased about 10 times compared with the original method. This method may also be applied to other EIAs of digitoxin, alpha-fetoprotein and thyroid-stimulating hormone.