An automated streamlined method for the therapeutic drug monitoring of digoxin based on the online-solid-phase extraction liquid chromatography tandem high-resolution mass spectrometry.

RATIONALE: Digoxin is widely used in the clinical treatment of cardiovascular diseases. However, due to its extremely narrow therapeutic window, therapeutic drug monitoring (TDM) is vitally important. In consideration of the time-consuming and labor-intensive nature of the traditional techniques, an automated and efficient method was required for the clinical individualized TDM of digoxin. METHODS: An online solid-phase extraction liquid chromatography tandem high-resolution mass spectrometry (online-SPE-LC-HRMS) method was developed and applied for the determination of digoxin in plasma. The online SPE-LC steps included pretreatment and separation of plasma samples that were carried out using a Waters Oasis HLB cartridge and XBridge Shield RP18 column, respectively. A high-resolution Q Orbitrap mass spectrometer with targeted-selected ion monitoring in negative scan mode was applied to monitor formate-adduct ions [M + HCOO]- m/z 825.42781 for digoxin. RESULTS: Linearity was shown over the range 0.1-10 ng mL-1 for digoxin with correlation coefficients of R2 > 0.999. The lower limit of quantitation (LLOQ) for digoxin was 0.1 ng mL-1 . Extraction recoveries ranged from 82.61% to 94.28% for digoxin. The intra- and inter-day precision values were < 5.53% with accuracy ranging from 84.97% to 96.75%. The total running time was 10 min for each sample. CONCLUSION: The established method displayed satisfactory recoveries, accuracy, precision, and stability, and successfully applied on the TDM of digoxin. This automated streamlined method provides a powerful tool to guide the individualized administration of digoxin, which is significant for the practice of precision medicine.

An electrochemical immunosensor for digoxin using core-shell gold coated magnetic nanoparticles as labels.

A simple, sensitive, and low-cost immunosensor was designed for the detection of digoxin through core-shell gold coated magnetic nanoparticles (Fe3O4-Au-NPs) as an electrochemical label. Having had such a large potential for a variety of applications, Fe3O4-Au-NPs have attracted a considerable attention and are actively investigated recently. Digoxin is a cardiac glycoside which, at high level, can indicate an increased risk of toxicity. This new competitive electrochemical immunosensor was developed based on antigen-antibody reaction employing antigen (Ag) labeled Fe3O4-Au-NPs and PVA modified screen-printed carbon electrode surface in order to detect the serum digoxin. The structures of Fe3O4-Au-NPs were studied by transmission electron microscopy, X-ray diffraction and Fourier transformed infrared spectroscopy. Cyclic voltammetry and differential pulse voltammetry (DPV) were employed to determine the physicochemical and electrochemical properties of immunosensor. DPV was employed for quantitative detection of digoxin in biological samples. The developed immunosensor was capable to detect digoxin in the range from 0.5 to 5 ng mL(-1), with a detection limit as low as 0.05 ng mL(-1). The proposed method represented acceptable reproducibility, stability, and reliability for the rapid detection of digoxin in serum samples.

Identification of multiple ingredients for a Traditional Chinese Medicine preparation (bu-yang-huan-wu-tang) by liquid chromatography coupled with tandem mass spectrometry.

Bu-yang-huan-wu-tang (BYHWT) is a popular Traditional Chinese Medicine formula consisting of seven herbal medicines (Astragalus membranaceus, Angelica sinensis, Paeonia lactiflora, Ligusticum chuanxiong, Carthamus tinctorius, Amygdalus persica and Pheretima aspergillum), that has been used in China for centuries to overcome stroke-induced disability. To ensure the consistency of quality, a reliable analytical method is required, therefore, we developed a liquid chromatography with tandem mass spectrometry (LC-MS/MS) method for quantitative analysis of the major constituents in BYHWT. The herbal ingredients consisting of the cycloartane-type triterpene glycosides of astragaloside I, astragaloside II and astragaloside IV; isoflavones of formononetin, ononin calycosin, calycosin-7-O-ß-d-glucoside; ligustilide and paeoniflorin were separated on a C18 column with gradient elution of methanol/10 mM ammonium acetate buffer-formic acid (100:0.1, v/v). This study was performed by a mass spectrometer using electrospray ionization (ESI) with positive ionization ions monitored in the multiple reaction-monitoring (MRM) mode. The linearity, accuracy, precision, limit of detection (LOD) and lower limit of quantification (LLOQ) were validated for this quantification method, and the sensitivity, reliability and reproducibility were all confirmed. The experiments provided a good method for analyzing BYHWT extracts. This study also quantitated the active components in various brands of commercially available products. The results indicated that the pharmaceutical industrial products of BYHWT exhibited considerable variation in their contents of the herbal compounds.

Nanomagnet-based removal of lead and digoxin from living rats.

In a number of clinical conditions such as intoxication, bacteraemia or autoimmune diseases the removal of the disease-causing factor from blood would be the most direct cure. However, physicochemical characteristics of the target compounds limit the applicability of classical filtration and diffusion-based processes. In this work, we present a first in vivo magnetic blood purification rodent animal model and demonstrate its ability to rapidly clear toxins from blood circulation using two model toxins with stable plasma levels (lead (Pb(2+)) and digoxin). Ultra-strong functionalized metal nanomagnets are employed to eliminate the toxin from whole blood in an extracorporeal circuit. In the present experimental demonstration over 40% of the toxin (i.e. lead or digoxin) was removed within the first 10 minutes and over 75% within 40 minutes. After capturing the target substance, a magnetic trap prevents the toxin-loaded nanoparticles from entering the blood circulation. Elemental analysis and magnetic hysteresis measurements confirm full particle recovery by simple magnetic separation (residual particle concentration below 1 µg mL(-1) (detection limit)). We demonstrate that magnetic separation-based blood purification offers rapid blood cleaning from noxious agents, germs or other deleterious materials with relevance to a number of clinical conditions. Based on this new approach, current blood purification technologies can be extended to efficiently remove disease-causing factors, e.g. overdosed drugs, bacteria or cancer cells without being limited by filter cut-offs or column surface saturation.

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.

Device for continuous extracorporeal blood purification using target-specific metal nanomagnets.

BACKGROUND: The present work illustrates how magnetic separation-based blood purification using ultra-strong iron nanomagnets can be implemented into an extracorporeal blood purification circuit. By this promising technique, today's blood purification may be extended to specifically filter high-molecular compounds without being limited by filter cut-offs or column surface saturation. METHODS: Blood spiked with digoxin (small molecule drug) and interleukin-1ß (inflammatory protein) was circulated ex vivo through a device composed of approved blood transfusion lines. Target-specific nanomagnets were continuously injected and subsequently recovered with the aid of a magnetic separator before recirculating the blood. RESULTS: Magnetic blood purification was successfully carried out under flow conditions: already in single-pass experiments, removal efficiencies reached values of 75 and 40% for digoxin and interleukin-1ß, respectively. Circulating 0.5 L of digoxin-intoxicated blood in a closed loop, digoxin concentration was decreased from initially toxic to therapeutic concentrations within 30 min and purification extents of 90% were achieved after 1.5 h. CONCLUSIONS: Magnetic separation can be successfully implemented into an extracorporeal blood purification device. Simultaneous and specific filtering of high-molecular compounds may offer promising new therapeutic tools for the future treatment of complex diseases, such as sepsis and autoimmune disorders.

[Preparation of biologically transformed raw material from woolly foxglove Digitalis lanata Ehrh and isolation of digoxin from it]. / Poluchenie biotransformirovannogo syr'ia naperstianki sherstistoi Digitalis lanata Ehrh i vydelenie iz nego digoksina.

A biotechnological approach is proposed for conservation of a terraneous part of woolly foxglove under anaerobic conditions with a subsequent air-sun drying of the biologically transformed raw material. During the conservation primary foxglove glycosides completely convert to secondary ones which do not transform further. A simple method is described for preparation from the transformed raw material of an enriched glycoside fraction with the yield of 3.6% and for isolation from this fraction of highly purified digoxin with the yield of 0.06% of the starting raw material, and the other secondary glycosides can be also isolated.

Effect of canrenone on the digitalis site of Na+/K(+)-ATPase in human placental membranes and in erythrocytes.

It has been reported that canrenone, which is used in hypertensive therapy as an antialdosteronic drug, may also act as a blocker of ouabain effects. Several studies suggest that human plasma contains an endogenous ouabain-like factor similar to ouabain, which may be increased in hypertension, in pregnancy, and in the neonatal state. This study evaluated (1) the effect of canrenone on Na+/K(+)-ATPase in relation to ouabain in human placental membranes and erythrocytes by 3H-ouabain binding assay; (2) the capacity of canrenone (10 microM) to reverse the inhibition of Na+/K(+)-ATPase by ouabain and by ouabain-like factor (from umbilical cord plasma) in human erythrocytes employing a 86Rb uptake assay. Increasing concentrations of canrenone (0-350 microM) partially competed with 3H-ouabain binding in placental membrane (40%) and erythrocytes (60%). Scatchard plot from radioreceptor assay in placental membrane showed that ouabain and canrenone compete for the same binding site. In erythrocytes, canrenone completely reversed the inhibition caused by ouabain (5 x 10(-9) M) and ouabain-like factor (2 x 10(-9) M ouabain equivalents). A reduction of inhibition of about 50% was observed with ouabain and ouabain-like factor respectively at a concentration of 5 x 10(-8) M and 2 x 10(-8) M (ouabain equivalents). Our results thus provide evidence that canrenone, at therapeutical concentrations, is a partial competitive agonist of ouabain and of ouabain-like factor in human placental membranes and erythrocytes.

Heterogeneous post-column immunoreaction detection using magnetized beads and a laboratory-constructed electromagnetic separator.

The nature of immune reactors allows development of quantitative analytical methods that are highly selective and can often be used directly with complex biological matrixes such as blood, plasma or urine. A major limitation of immunoassay is that antibodies are sometimes unable to discriminate structurally similar species such as drug metabolites and synthetic analogs. The problem associated with the lack of discrimination can be circumvented by coupling immunoassay with liquid chromatography post-column. The most commonly used separation method in post-column immunoreaction detection is the affinity column. Affinity columns may create undesired effects such as a compromise of the chromatographic separation efficiency, the requirement for an antibody with fast reaction kinetics and the need for flushing the column. This paper reports a post-column immunoreaction detection system coupled with a laboratory-constructed on-line magnetic separation flow chamber that is designed to overcome these problems. The system uses disposable magnetic beads as a solid-phase support for separation that can be easily removed from the system. The model analytes chosen for this study were digoxin and its metabolites due to the commercial availability of monoclonal antibodies for these compounds. Digoxin was separated using a chromatographic method prior to being interfaced through a liquid handler system to the immunoreactor. Compatibility of the HPLC mobile phase was determined to be acceptable with a mixing ratio of 1:3 between the LC fraction and immunoreagent solution. The dynamic range of the calibration curve in digoxin-spiked phosphate buffer was found to be 0.25-12 ng/ml and a quadratic fit was found to provide the best fit to the data with a correlation coefficient of 0.9974. The residual error for all standards was less than 15%. The percentage RSDs for the two controls, 2 and 10 ng/ml, were 6.88 and 4.82% (n = 6) and the percentage errors were 7.07 and -6.89% (n = 6), respectively.

Applicability of mass spectrometry to detect coeluting impurities in high-performance liquid chromatography.

An array of pharmaceutical compounds and impurities were used to investigate the applicability of atmospheric pressure ionization mass spectrometry (MS) to routinely detect coeluting impurities in HPLC (i.e. peak purity). Four drugs were individually tested against their related impurity set using a straightforward HPLC-MS peak purity strategy. For the investigated set, which represents 24 unique drug-impurity permutations, 75% of the coeluting impurities were detected at levels <1.0%, including one-third at 0.1% (%, w/w). Factors that affect the applicability of this peak purity approach are also discussed.

Immunoaffinity screening with capillary electrochromatography.

Highly efficient capillary electrochromatographic separations of cardiac glycosides and other steroids are presented. Employing butyl-derivatized silica particles as stationary phase resulted in a nearly three times faster electroosmotic flow (EOF) compared to capillary electrochromatography (CEC) with octadecyl silica particles. On-column focusing with a preconcentration factor of 180 was performed and separation efficiencies of up to 240,000 plates per meter were obtained. Using label-free standard UV absorbance, detection limits of 10-80 nM were reached for all steroids tested. For screening of cardiac glycosides, e.g., digoxin and digitoxin in mixtures of steroids, CEC was combined with immunoaffinity extraction using immobilized polyclonal anti-digoxigenin antibodies and F(ab) fragments. Simply adding small amounts of antibody carrying particles to the samples and comparing chromatograms before and after antibody addition allowed screening for high affinity antigens in mixtures with moderate numbers of compounds. Under conditions of competing antigens, affinity fingerprints of immobilized anti-digoxigenin and anti-digitoxin antibodies were obtained, reflecting the cross-reactivity of eleven steroids. The method provides high selectivity due to the combination of bioaffinity interaction with highly efficient CEC separation and UV detection at several wavelengths in parallel. This selectivity was exploited for the detection of four cardiac glycosides in submicromolar concentrations in an untreated urine sample.

Removal of digoxin by column for specific adsorption of beta(2)-microglobulin: a potential use for digoxin intoxication.

BACKGROUND: A beta(2)-microglobulin adsorption column used for the treatment of dialysis-related amyloidosis removes serum beta(2)-microglobulin by recognition of lipophilic residue in the protein. No data are available for the adsorption of the highly lipophilic drug digoxin. METHODS: In vivo clearance of digoxin with the beta(2)-microglobulin column was measured by a single use of the column in 8 patients receiving hemodialysis with a therapeutic level of digoxin. In vitro adsorption was evaluated by use of incubation with adsorbent of the column and digoxin or ranitidine, a hydrophilic drug. Clearance with the beta(2)-microglobulin column was further compared with that obtained by use of activated charcoal in the dogs intoxicated with digoxin. RESULTS: Digoxin concentration was reduced from 1.11 +/- 0.25 ng/mL to 0.57 +/- 0.15 ng/mL at 240 minutes after initiation of hemoperfusion with the column in the patients. Digoxin clearance with the beta(2)-microglobulin column was about 145 +/- 20 mL/min, with a blood flow rate of 160 to 220 mL/min (80% of plasma flow rate). Eighty-five percent of digoxin was adsorbed in vitro, and the capacity of the beta(2)-microglobulin column was not saturated until a toxic level was reached (50 ng/mL). This value was higher than that obtained with use of charcoal. In dogs with digoxin intoxication, digoxin clearance was 38.9 +/- 1.5 mL/min, with a blood flow rate of 50 mL/min (95% of plasma flow rate), which was almost twice as that achieved with charcoal. The degree of thrombocytopenia and leukopenia was small with use of the beta(2)-microglobulin column. CONCLUSION: These data suggested that the beta(2)-microglobulin column selectively adsorbs digoxin. This column is a promising tool for the treatment of digoxin intoxication, especially in patients undergoing hemodialysis.

[Separation and purification of an endogenous inhibitor of sodium pump from chansu by thin-layer chromatography and reversed-phase high performance liquid chromatography].

An endogenous inhibitor of the sodium pump from the Chinese medication Chansu was purified. The dry substance Chansu was extracted with methanol. The dry residue dissolved in water and filtered subsequently through membrane filters with the exclusion size of 1000 Da, 3000 Da and 10000 Da in a Filtron Pro Vario-3-System and applied to thin-layer chromatographic plate made of Silica gel 60 F254 + 366 developed with a mixture of CHCl3-MeOH-H2O(75:20:5, volume ratio). The fractions with Rf 0.55 inhibiting the sodium pump were purified on an HPLC C18-RP column using a linear H2O-methanol gradient with 220 nm and 300 nm DAD detection. The bioactivity was measured by 86Rb-uptake into human red blood cells. The results showed that a low molecular weight, water soluble compound, which inhibited the sodium pump activity in the red blood cells and had a maximum absorbance at 250 nm was isolated from the Chinese medication Chansu. Several mg of the compound in pure state could be obtained from 1 kg Chansu. It was different from ouabain and proscillaridin A in chemical structure, because ouabain and proscillaridin A show a UV maximum absorption at 220 nm and 300 nm, while the new inhibitor at 250 nm.

A novel high-pressure liquid-liquid extraction process for downstream processing in biotechnology: extraction of cardiac glycosides.

This investigation examines phase equilibrium phenomena that can be used to create two water-like solvents for liquid-liquid extraction in downstream processing in biotechnology: a completely miscible, binary liquid mixture of water and a hydrophilic organic solvent (e. g., an alcohol) reveals a liquid phase split, when it is pressurized with a "near-critical" gas (i.e., a substance which at ambient conditions is a gas, near its critical temperature). This phase split results in two hydrophilic liquid phases. Making use of this phenomenon in process development first requires research on the phase split phenomenon and, second, research on the feasibility of biomolecule extraction and separation. In this study, basic fluid phase equilibrium phenomena are briefly described. Then, experimental results are reported for the partitioning of small amounts of cardiac glycosides (digitoxin and digoxin) on coexisting liquid phases in the high-pressure, three-phase, vapor-liquid-liquid equilibrium of the ternary system of "near critical" CO(2) + water + 1-propanol, at 313 K and 333 K. Finally, a process for extraction and separation of the aforementioned glycosides by means of the high-pressure phase equilibrium phenomenon is discussed.

[Solubility and dissolution rate of digoxin from Digitalis lanata drug extracts] . / Lösungsverhalten von Digoxin in Gegenwart pflanzlicher Begleitstoffe aus Digitalis-lanata-Trockenextrakten. Teil 1: Löslichkeit und Lösungsgeschwindigkeit.

The influence on solubility and dissolution rate was investigated for digoxin as a model drug with a very low solubility in water. The investigations were carried out with different fractions of extracts from leaves of Digitalis lanata. These fractions differ in the composition of concomitant compounds. The solubility of digoxin from the extract fractions is increased up to 42 times, with considerable differences between the fractions. The solubility depends on the weight of the extract fraction; a limit of solubility exists. Even after separation of the solved extract components the solubility of digoxin in the residues is larger than that of the pure digoxin. The dissolution rate of digoxin of "Vorgereinigter Gesamtglykosidextrakt (VE)" and the glycosid fraction G 1 is influenced significantly, whereas digoxin in the glycosid fraction G 4 has such a degree of purity that the solubility properties are not influenced by the small amount of concomitant compounds. After 10 min already 50.4% of the digoxin in the extract fraction G 1 are dissolved, while only 21.7% of the pure digoxin are dissolved in that interval. The extract fractions exhibit different wettability properties, so that the increased dissolution rate could be attributed to improved wettability of the extract fractions. Physical mixtures of crystal-line digoxin and compounds of the extracts of the almost digoxin free fraction G 2 did not exert an influence on the dissolution behavior. Different batches of the extract fractions showed different solubility in spite of comparable digoxin content.

Extraction and quantitation of digoxin and acetyldigoxin from the Digitalis lanata leaf via near-supercritical methanol-modified carbon dioxide.

An extraction process is reported that employs a near-supercritical mixture of CO2 and MeOH to extract the cardiac glycoside, digoxin, from the Digitalis lanata leaf. The method development of the sample preparation procedure is presented in detail, and reasons for trends that occur in the natural products extraction are given.

Identification and preliminary characterization of two human digitalis-like substances that are structurally related to digoxin and ouabain.

In order to characterize the structure of endogenous digitalis-like immunoreactive factor (DLIF), we utilized peritoneal dialysis fluid from patients with chronic renal failure as a source of endogenous digitalis-like immunoreactive factor (DLIF), and subjected it to one-step ion exchange chromatography, followed by one step reverse HPLC. Crude dialysis fluid contained 0.09 ng/ml of DLIF, and using Amberlite XAD-2 chromatography we extracted 110 ng of DLIF from 800 ml of dialysis fluid. By applying this partially purified DLIF to our HPLC system, we discerned three peaks of DLIF activity, with retention times of 34, 58 and 63 minutes. The first peak overlapped the elution profile of ouabain, and the third peak co-eluted precisely with digoxin. The second DLIF peak was not in proximity to any of the digitalis-like markers employed. Thus, our results indicate that DLIF isolated from peritoneal dialysis fluid exists in three distinct forms, one of which resembles ouabain, and one which is identical to digoxin.

Partial purification of endogenous digitalis-like compound(s) in cord blood.

Increasing evidence indicates the presence of endogenous digitalis-like compound(s) in human body fluids. In this preliminary report, we describe a study of the partial purification by HPLC of these compounds in the plasma of neonates (who have particularly high concentrations of this substance) and adults. Plasma samples from neonates (cord blood) and adults, lyophilized and extracted with methanol, were applied on a 300 x 3.9 mm C18 Nova Pak column and eluted with a mobile phase of acetonitrile/methanol/water (17/17/66 or 14/14/72 by vol) and, after 30 min, with 100% methanol. We assayed eluted fractions for inhibitory activity of 86Rb uptake and for digoxin-like immunoreactivity. The elution profile revealed a first peak of inhibitory activity of 86Rb uptake at the beginning of the chromatography; another peak was eluted with the 100% methanol. The two peaks also cross-reacted with antidigoxin antibodies. Because the second peak could possibly reflect the nonspecific interference of various lipophilic compounds, we focused our attention on the first peak. For these fractions dose-response curves for 86Rb uptake and for displacement of digoxin were parallel, respectively, to those of ouabain and digoxin, suggesting similarities of digoxin-like immunoreactive substance to cardiac glycosides. Similar chromatographic profiles were also obtained for plasma from adults, suggesting that the endogenous glycoside-like compound(s) in the neonate may be the same as those in the adult.