Chromato-photocolorimetric determination of tselanid (lanatoside C)

Methods have been developed for the chromato-colorimetric determination of tselanid (lanatoside C) as such and in tablets and solutions. The chloroform-methanol-water (80:19:1) solvent system was used. Chromatography was performed by the ascending method on Silufol UV-254 plates. The revealing agent was a 1% solution of vanillin in 10% perchloric acid. Quantitative determination was performed photocolorimetrically by the reaction with sodium picrate. The methods developed have given accurate results correlating with those of high-performance liquid chromatography and the biological method. The relative error of this determination does not exceed ±4% [1].Tashkent Pharmaceutical Institute. All-Union Scientific-Research Institute of Pharmacy, Moscow. Translated from Khimiya Priodnykh Soedinenii, No. 3, pp. 332–336, May–June, 1986.     

The specific fluorimetric determination of digoxin.

The simultaneous determination of Cd, Cu, Pb and Zn in lead and zinc concentrates by fundamental, second-harmonic and linear-sweep a.c. and pulse polarographic methods is described. Calibration curves are linear over wide concentration ranges, so that both major and minor trace constituents can be determined in the same experiment; thus the polarographic method is highly competitive with atomic absorption spectrometry (a.a.s.). Conventional a.c. polarography and a.a.s. were compared in the first instance with conservative instrumentation. More sophisticated polarographic methods were then utilized; with the phase-selective linear sweep a.c. (fundamental- and second-harmonic) methods the four elements were determined simultaneously from voltammograms obtained in less than 20 s down to the 10^-6-10^-7M concentration range.     

Permanently oriented antibody immobilization for digoxin determination with a flow-through fluoroimmunosensor

This paper reports a new flow-through fluoroimmunosensor, the function of which is based on antibodies immobilized on an inmunoreactor of controlled-pore glass (CPG), for determination of digoxin, used in the treatment of congestive heart failure and artery disease. The immunosensor has a detection limit of 1.20 microg L(-1) and provides high reproducibility (RSD=4.5% for a concentration of 0.0025 mg L(-1), and RSD=6.7% for 0.01 mg L(-1)). The optimum working concentration range was found to be 1.2 x 10(-3)-4.0 x 10(-2) mg L(-1). The lifetime of the immunosensor was about 50 immunoassays; if stored unused its lifetime can be extended to three months. A sample speed of about 10-12 samples per hour can be attained. Possible interference from substances with structures similar to digoxin (morphine, heroin, tebaine, codeine, pentazocine and narcotine) was investigated. No cross-reactivity was seen at the highest digoxin: interferent ratio studied (1:100). The proposed fluoroimmunosensor was successfully used to determine digoxin concentrations in human serum samples.     

Micro high-performance liquid chromatographic determination of cardiac glycosides in beta-methyldigoxin and digoxin tablets

A micro high-performance liquid chromatographic (micro-HPLC) method has been developed for the assay of beta-methyldigoxin and digoxin tablets. Quantitation of cardiac glycosides in tablets was carried out by the incorporation of dexamethasone as an internal standard. The procedure consisted of disintegration of tablets, extraction with acetone-ethanol (9:1) and injection for micro-HPLC on an ODS micro column, using acetonitrile-water (28:72) for beta-methyldigoxin tablets and methanol-water (1:1) for digoxin tablets; the effluent was monitored by UV detection at 220 nm. The average values of the contents in beta-methyldigoxin and digoxin tablets were 99.6 and 100.2% of the labelled amounts, respectively. The proposed method is sufficiently precise and sensitive to examine the content uniformity of tablets.     

Simultaneous determination of digoxin and digitoxin by micellar electrokinetic chromatography and application to drug formulations

A simple micellar electrokinetic chromatographic method is described for simultaneous determination of digoxin and digitoxin. The simultaneous analysis of digoxin and digitoxin was performed in Tris buffer (10 mM; pH 9) with 90 mM sodium dodecyl sulfate and 10% isopropyl alcohol as an anionic surfactant and organic modifier. Under these conditions, good separation with high efficiency is achieved in short analysis times. Several parameters affecting the separation of the drugs were studied, including the pH and concentrations of the Tris buffer and sodium dodecyl sulfate. The linear range of the method for the determination of digoxin and digitoxin was over 0.01–0.3 mg/mL; the detection limit (signal to noise ratio = 3; injection 3.5 kPa 3 s) was 4 and 6 μg/mL, respectively. Application of the proposed method to the determination of digoxin in commercial tablets and in injections proved to be feasible.     

Molecularly imprinted SPE and MEKC with in-capillary sample preconcentration for the determination of digoxin in human urine

Molecularly imprinted solid-phase extraction (MISPE) combined with MEKC was used for clean-up, preconcentration and determination of digoxin in the presence of its aglycon digoxin (digoxigenin) in human urine samples. In addition, the use of an in-capillary sample concentration electrophoretic technique by sweeping was investigated to enhance the concentration sensitivity in MEKC. The highly selective, fast and effective sample pretreatment by MISPE along with the preconcentration by sweeping could overcome the low sensitivity of the highly efficient capillary electrophoresis separation with UV detection. The optimization of the variables affecting the separation as well as MISPE conditions procedure was carried out to select the best conditions of selectivity and sensitivity to determine digoxin at low concentration levels in urine. To demonstrate the suitability of the developed method several analytical characteristics (selectivity, linearity, accuracy, precision, and LOD) were evaluated. Satisfactory results were obtained in terms of linearity (r > 0.99), recovery (95.4-96.5% with RSD from 1.3% to 2.6%), precision (RSD from 0.3% to 1.7% for migration times and from 2.1% to 7.3% for corrected peak areas), and sensitivity (LODs of 6 μg/L with 5 mL of sample or 1.2 μg/L with 25 mL). The proposed MISPE-MEKC method was satisfactorily applied to the analysis of spiked human urine samples achieving a concentration factor up to 7500-fold.     

A MIP-based flow-through fluoroimmunosensor as an alternative to immunosensors for the determination of digoxin in serum samples

This work reports a comparative study of two automated flow-through fluorosensors for the determination of digoxin in serum samples: an immunosensor with an anti-digoxin polyclonal antibody as the reactive phase permanently immobilised on controlled-pore glass and a sensor with a selective reaction system based on a methacrylic molecularly imprinted polymer (MIP) synthesised by bulk polymerisation. The variables affecting the sensitivity and dynamic range of the sensors (e.g. the carrier and elution solutions, flow rates, pH and reagent concentrations) were optimized, and the binding characteristics of their reactive phases were compared in a competitive fluorescent assay. Digoxin was reproducibly determined by both sensors at the milligram per litre level (detection limit = 1.20 × 10⁻³ mg L⁻¹ and RSD = 4–7% for the immunosensor; detection limit = 1.7 × 10⁻⁵ mg L⁻¹ and RSD = 1–2% for the MIP sensor). No cross-reactivity with digoxin-related compounds was seen for either sensor at a digoxin/interferent ratio of 1:100. The lifetime of the immunosensor was about 50 immunoassays; its shelf life, when unused, is about 3 months. The lifetime of the MIP sensor was over 18 months. Both sensors were used to determine the digoxin concentration of human serum samples with satisfactory results.     

Determination of digoxin in human-serum

Summary Four immunological assays (RIA, ELISA, EMIT, FPIA) for digoxin were characterized with respect to reproducibility, detection limit, selectivity, and accuracy, followed by the comparison with HPLC. Afterwards the serum samples of nearly 60 patients were analyzed by these five methods.It could be shown, that the reproducibility at 2 g/l was fairly good for all methods. Reliable analysis in the lower concentration range (< 1 g/l) was difficult with two assays, because of insufficient detection limits. There was a marked cross-reactivity to other digitalis glycosides. Moreover, correlation between the immunological methods and in comparison to the HPLC-method was low.

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Bestimmung von Digoxin in HumanserumVergleich einiger immunologischer Assays mit einer vorgeschlagenen HPLC-Referenzmethode

     

An optical sensor for the determination of digoxin in serum samples based on a molecularly imprinted polymer membrane

This paper reports the synthesis and testing of a molecularly imprinted polymer membrane for digoxin analysis. Digoxin-specific bulk polymer was obtained by the UV initiated co-polymerisation of methacrylic acid and ethylene glycol dimethacrylate in acetonitrile as porogen. After extracting the template analyte, the ground polymer particles were mixed with plasticizer polyvinyl chloride to form a MIP membrane. A reference polymer membrane was prepared from the same mixture of monomers but with no template. The resultant membrane morphologies were examined by scanning electron microscopy. The imprinted membrane was tested as the recognition element in a digoxin-sensitive fluorescence sensor; sensor response was measured using standard solutions of digoxin at concentrations of up to 4 × 10⁻³ mg L⁻¹. The detection limit was 3.17 × 10⁻⁵ mg L⁻¹. Within- and between-day relative standard deviations RSD (n = 5) were in the range 4.5-5.5% and 5.5-6.5% respectively for 0 and 1 × 10⁻³ mg L⁻¹ digoxin concentrations. A selectivity study showed that compounds of similar structure to digoxin did not significantly interfere with detection for interferent concentrations at 10, 30 and 100 times higher than the digoxin concentration. This simply manufactured MIP membrane showed good recognition characteristics, a high affinity for digoxin, and provided satisfactory results in analyses of this analyte in human serum.     

Separation techniques in radioimmunoassays for digoxin and insulin

Ohne Zusammenfassung

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Separation techniques in radioimmunoassays for digoxin and insulin

     

The development of a fully automated solid-phase radioimmunoassay for digoxin

A radioimmunoassay for using antibody coated tubes and a unique radiotracer is described. The assay was designed specifically for use with the fully automated instrument Micromedic Systems «Concept 4 Automatic Radioassay». Antisera to digoxigenin-3-succinyl-BSA were raised in rabbits, purified by ammonium sulfate precipitation, and coated onto polypropylene tubes. An iodinated histamine derivative of digoxigenin which is insensitive to serum variability is utilized. The standard curve is linear for digoxin concentrations between 0.4 and 6.4 ng/ml and is parallel for two different sample sizes. Recovery of added digoxin was quantitative. Sample values obtained by this method correlate well with values obtained by other methods. The assay can either be performed manually or fully automated on Concept 4. The advantages provided by total automation are discussed.     

In vitro selection and characterization of deoxyribonucleic acid aptamers for digoxin

The low therapeutic index of digoxin necessitates careful monitoring of its serum levels. Most of digoxin immunoassays suffer from interferences with digoxin-like immunoreactive substances. Since aptamers have been shown to be highly specific for their targets, the aim of this study was to develop DNA aptamers for this widely used cardiac glycoside. Digoxin was coated onto the surface of streptavidin magnetic beads. DNA aptamers against digoxin were designed using Systematic Evolution of Ligands by Exponential enrichment method (SELEX) by 11 iterative rounds of incubation of digoxin-coated streptavidin magnetic beads with synthetic DNA library, DNA elution, electrophoresis and PCR amplification. The PCR product was cloned and sequenced. Binding affinity was determined using digoxin–BSA conjugate, coated onto ELISA plate. Inhibitory effect of anti-digoxin aptamer was conducted using isolated guinea-pig atrium. Three aptamers (D1, D2 and D3) were identified. Binding studies of fluorescein-labeled truncated (without primer binding region) D1 and D2 and full length D1 anti-digoxin aptamers were performed and their corresponding dissociation constants values were 8.2 × 10⁻⁹, 44.0 × 10⁻⁹ and 17.8 × 10⁻⁹ M, respectively. This is comparable to what other workers have obtained for interaction of monoclonal antibodies raised against digoxin. There was little difference in binding affinity between full length and truncated anti-digoxin D1 aptamer. D1 anti-digoxin aptamer also inhibited the effects of digoxin on the isolated guinea-pig atrium. D1 anti-digoxin aptamer distinguished between digoxin and ouabain in both tissue study and binding experiments. Our finding indicated that D1 anti-digoxin aptamer can selectively bind to digoxin. Further studies might show its suitability for use in digoxin assays and as a therapeutic agent in life-threatening digoxin toxicity.     

Unexpected behavior of digoxin at elevated temperatures in reversed-phase liquid chromatography

Summary Increased retention of digoxin has been observed at elevated temperatures on both 10 nm and 30 nm porediamter, RP-18 packing. This result is the opposite effect compared with the decreased retention under the same conditions with digoxin aglycon-digoxigenin. Rotation around the C−C σ-bonds in the digoxin molecule is presumed; the rod-like molecules of the newlyobtained digoxin penetrate stationary phase pores more easily thus increasing retention.     

Development of a high-performance liquid chromatographic-post-column fluorogenic assay for digoxin in serum

A quantitative, sensitive and specific assay for digoxin was developed using a high-performance liquid chromatographic (HPLC) system with post-column (PC) fluorogenic derivatization. Separation of digoxin from its metabolites was accomplished using a 15 cm X 4.6 mm I.D., 3-microns octadecylsilyl HPLC column and an optimum mobile phase of methanol-ethanol-isopropanol-dehydroascorbic acid (52:3:1:45, v/v). Concentrated hydrochloric acid, used as the PC derivatization reagent, was delivered by hexane displacement from a polyvinyl chloride pressure vessel. Construction of the pressure vessel is described. The mixture of HPLC effluent and PC reagent was passed into a 20-m knitted reactor (PTFE tubing) maintained at 79.0 +/- 0.2 degrees C. The resultant fluorophores were monitored by a fluorescence detector equipped with a 360-nm excitation filter and a 425-nm emission filter. Specificity of this HPLC-PC assay for digoxin in the presence of its metabolites was demonstrated. Also, numerous steroids evaluated did not produce fluorescence under these conditions. An extraction procedure for evaluating digoxin in serum without interference from endogenous compounds was also developed. Detector response to digoxin was linear from 0.5 to 3.3 ng extracted from serum.     

On the influence of hyperlipoproteinemia on the results of insulin and digoxin radioimmunoassays

Conclusions From the current results no appreciable influence of triacylglycerol (up to 10 mmol/serum) and cholesterol (up to 14 mmol/l serum) on digoxin and insulin radioimmunoassays with different separation techniques could be detected. Therefore it has been concluded that hyperlipoproteinemic serum samples with lipid concentrations up to these limits did not deteriorate quality control parameters.

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Zum Einfluß von Hyperlipoproteinämien auf die Ergebnisse radioimmunologischer Insulin- und Digoxin-Bestimmungen