Crataegus mexicana (Tejocote) Exposure Associated with Cardiotoxicity and a Falsely Elevated Digoxin Level.

INTRODUCTION: A species of hawthorn, Crataegus mexicana (tejocote), has been marketed as a weight-loss supplement that is readily available for purchase online. While several hawthorn species have shown clinical benefit in the treatment of heart failure owing to their positive inotropic effects, little is known about hawthorn, and tejocote in particular, when consumed in excess. We describe a case of tejocote exposure from a weight-loss supplement resulting in severe cardiotoxicity. CASE REPORT: A healthy 16-year-old girl presented to an emergency department after ingesting eight pieces of her mother's tejocote root weight-loss supplement. At arrival, she was drowsy, had active vomiting and diarrhea, and had a heart rate of 57 with normal respirations. Her initial blood chemistries were unremarkable, except for an elevated digoxin assay of 0.7 ng/mL (therapeutic range 0.5-2.0 ng/mL). All other drug screens were negative. She later developed severe bradycardia and multiple episodes of hypopnea that prompted a transfer to our institution, a tertiary pediatric hospital. Her ECG demonstrated a heart rate of 38 and Mobitz type 1 second-degree heart block. She was subsequently given two vials of Digoxin Immune Fab due to severe bradycardia in the setting of suspected digoxin-like cardiotoxicity after discussion with the regional poison control center. No clinical improvement was observed. Approximately 29 hours after ingestion, subsequent ECGs demonstrated a return to normal sinus rhythm, and her symptoms resolved. DISCUSSION: Tejocote root toxicity may cause dysrhythmias and respiratory depression. Similar to other species of hawthorn, tejocote root may cross-react with some commercial digoxin assays, resulting in a falsely elevated level.

Taking Advantage of Assay Harmonization, Biotin Interference in the LOCI Digoxin Assay Could Be Eliminated by Using the ADVIA Centaur Digoxin Assay.

Biotin at elevated concentration interferes with immunoassays that utilize biotin in assay design. We earlier reported interference of biotin in the luminescent oxygen channeling assay (LOCI) digoxin assay which utilizes biotinylated antibody against digoxin. However, the ADVIA Centaur digoxin assay, also manufactured by Siemens Diagnostics, does not utilize biotin in assay design. We hypothesized that if the LOCI and the ADVIA Centaur digoxin assay are harmonized, then interference of biotin in the LOCI digoxin assay could be eliminated by using the ADVIA Centaur digoxin assay. We analyzed 25 specimens from patients receiving digoxin using both assays to investigate harmonization between these two assays. Then aliquots of drug-free serum pool were supplemented with various biotin concentrations (range: 10 ng/mL to 2000 ng/mL) followed by measuring apparent digoxin levels using the ADVIA Centaur digoxin assay. In another set of experiments, aliquots of a serum digoxin pool were supplemented with biotin (10-2000 ng/mL) and digoxin concentrations were measured by the ADVIA Centaur digoxin assay. We observed an excellent correlation between digoxin values obtained by the LOCI digoxin assay (reference method) and the ADVIA Centaur digoxin assay (y= 1.0514 x+0.1083, r=0.99) indicating that both assays are harmonized. We did not observe any interference of biotin even at a highly elevated concentration of 2000 ng/mL with the ADVIA Centaur digoxin assay. We conclude that taking advantage of assay harmonization, interference of biotin in the LOCI digoxin assay can be eliminated by using the ADVIA Centaur digoxin assay.

Rapid Detection of Drugs in Human Plasma Using a Small-Molecule-Linked Hybridization Chain Reaction.

Rapid detection and quantification of pharmaceutical drugs directly in human plasma is of major importance for the development of relevant point-of-care testing devices. Here, we report a method for detection and quantification of small molecules in human plasma. An assay employing a small molecule-linked hybridization chain reaction (HCR) has been devised for the detection of the pharmaceutically relevant drugs digoxin (Dig) and methotrexate (MTX). Double modification by small molecule ligands on the initiator strand act as sites to control the rate of the HCR. Upon protein binding to the modified initiator strand, the HCR is greatly inhibited. If the protein is preincubated with a sample containing the small molecule analyte, the protein binding site is occupied by the analyte and the initiator strand will initiate the HCR. This enables efficient detection and quantification of small-molecule analytes in nanomolar concentration even in 50% human plasma within 4 min. Thus, the rapidity and simplicity of this assay has potential for point-of-care testing.

Effect of green tea catechins on the pharmacokinetics of digoxin in humans.

BACKGROUND: Previous in vitro studies have reported the inhibitory effect of green tea on p-glycoprotein (p-gp) encoded by ABCB1. This study aimed to investigate the effect of green tea on the pharmacokinetics of digoxin, a typical probe drug of p-gp. METHODS: Sixteen healthy volunteers participated in this study. At Day 1, 0.5 mg of digoxin was administered via oral route. After a 14-day washout period, 630 mg of green tea catechins (GTC) was administered via oral route, followed by 0.5 mg of digoxin 1 hour later. From Day 16 through Day 28, 630 mg of GTC was administered alone. At Day 29, 630 mg of GTC and 0.5 mg of digoxin were administered in the same way as Day 15. Blood samples for the pharmacokinetic assessments of digoxin were collected up to 8 hours after each dose. Pharmacokinetic parameters were estimated by noncompartmental analysis. Area under the curve (AUC) and peak plasma concentration (Cmax) were compared using mixed effect model between digoxin alone and digoxin with GTC. ABCB1 was genotyped to determine whether its polymorphism affects digoxin-GTC interaction. RESULTS: Fifteen subjects completed the study. Compared to digoxin alone, the concomitant administration of digoxin and GTC significantly reduced the systemic exposure of digoxin: geometric mean ratios (GMR) and 90% confidence intervals (CI) of area under the concentration-time curve from time 0 to the last measurable time (AUClast) and Cmax were 0.69 (0.62-0.75) and 0.72 (0.61-0.85), respectively. The concomitant administration of digoxin and GTC following pretreatment of GTC (Day 29) similarly reduced the AUClast (GMR [90% CI]: 0.67 [0.61-0.74]) and Cmax (GMR [90% CI]: 0.74 [0.63-0.87]). In the comparison of the percentage changes from Day 1 (digoxin single administration) of AUClast between genotypes, C1236T variant type showed a significant difference to wild-type on Day 15 (concomitant administration of digoxin and GTC) (P=0.005). CONCLUSION: This study demonstrates that the coadministration of GTC reduces the systemic exposure of digoxin regardless of pretreatment of GTC.

Convallatoxin, the active cardiac glycoside of lily of the valley, minimally affects the ADVIA Centaur digoxin assay.

OBJECTIVE: Lily of the valley is a poisonous plant due to the presence of the cardiac glycoside convallatoxin which is known to interfere with serum digoxin measurement using the LOCI digoxin assay and other digoxin assays. We evaluated potential interference of convallatoxin as well as extract of lily of the valley with the ADVIA Centaur digoxin assay by comparing results obtained using the LOCI digoxin assay. MATERIALS AND METHODS: Aliquots of a drug-free serum pool and a digoxin serum pool were supplemented with nanograms to 1 µg quantities of convallatoxin or 1.0 and 2.5 µL of lily of the valley extract per milliliter of serum followed by measurement of digoxin concentrations using the LOCI and ADVIA Centaur digoxin assays. RESULTS: Apparent digoxin concentrations were minimal using the ADVIA Centaur digoxin assay when aliquots of drug-free serum were supplemented with convallatoxin or extract of lily of the valley but apparent digoxin levels were very high using the LOCI digoxin assay. Moreover, minimal interference in serum digoxin measurement using the ADVIA Centaur digoxin assay was observed when aliquots of serum digoxin pool were further supplemented with lily of the valley extract. As expected, the LOCI digoxin assay showed significant interference of convallatoxin in serum digoxin measurement. CONCLUSIONS: Significant interference of convallatoxin in serum digoxin measurement using the LOCI digoxin assay could be minimized using the ADVIA Centaur digoxin assay.

Biotin at High Concentration Interferes with the LOCI Digoxin Assay but the PETINIA Phenytoin Assay Is Not Affected.

Many automated immunoassays incorporate biotinylated antibodies and streptavidin-coated magnetic beads in the assay design. Biotin at elevated concentrations may interfere with these immunoassays. We evaluated potential interference of biotin on serum digoxin (LOCI assay utilizing biotinylated antibody) and phenytoin (PETINIA assay; no biotinylated antibody) measurements using the Vista 1500 analyzer. Aliquots of drug-free serum pool were supplemented with various biotin concentrations (range: 1 ng/mL to 250 ng/mL) followed by measuring apparent digoxin and phenytoin levels using appropriate immunoassays. In the second set of experiments, one serum pool was prepared from patients taking digoxin and another from patients taking phenytoin. Then aliquots of these serum pools were further supplemented with biotin followed by measuring digoxin or phenytoin concentrations. We observed apparent digoxin levels at 50 ng/mL biotin concentration or higher and also significant interference of biotin in serum digoxin measurement at a biotin concentration of 250 ng/mL. In contrast, we observed no interference of biotin in serum phenytoin measurement. We conclude that biotin interferes with the LOCI digoxin assay at a high concentration only.

Efficacy and effectiveness of anti-digoxin antibodies in chronic digoxin poisonings from the DORA study (ATOM-1).

CONTEXT: We hypothesized that in chronic digoxin toxicity, anti-digoxin antibodies (Fab) would be efficacious in binding digoxin, but this may not translate into improved clinical outcomes. OBJECTIVE: This study aims to investigate changes in free digoxin concentrations and clinical effects on heart rate and potassium concentrations in chronic digoxin poisoning when anti-digoxin Fab are given. MATERIALS AND METHODS: This is a prospective observational study. Patients were recruited if they have been treated with anti-digoxin Fab for chronic digoxin poisoning. Data was entered into a standardised prospective form, supplemented with medical records. Their serum or plasma was collected, analysed for free and bound digoxin and free anti-digoxin Fab concentrations. RESULTS: From September 2013 to February 2015, 36 patients (median age, 78 years; 22 females) were recruited from 18 hospitals. Median heart rate (HR) was 49 beats/min. Initial median digoxin and potassium concentrations were 4.7 nmol/L (3.6 µg/L) (range: 2.3-11.2 nmol/L) and 5.3 mmol/L (range: 2.9-9.2 mmol/L) respectively. Beta-blockers (n = 18), calcium antagonists (n = 6), spironolactone and/or angiotensin blocking agents (n = 24) were also used concomitantly. Renal impairment and gastrointestinal symptoms were present in 31 (86%) and 22 (63%) patients respectively. Five patients died from conditions unrelated to digoxin toxicity. Median change in HR was 8 beats/min post-Fab with no effect on blood pressure; they were 4, 10 and 17 beats/min for the 1, 2 and ≥3 vials of anti-digoxin Fab groups respectively. Concomitant treatments with potassium lowering agents (12/36) and inotropic drugs (7/36) were used. Gastrointestinal effects resolved in all 22 patients. The median decrease for potassium was 0.3 mmol/L. Digoxin concentration reduced from 3.8 to 0 nmol/L post-Fab. There was a rebound observed in the free digoxin concentration in 25 patients but none had associated clinical deterioration. CONCLUSIONS: One to two vials of anti-digoxin Fab initially bound all free digoxin confirming Fab efficacy. However, this was associated with only a moderate improvement in HR and potassium, suggesting bradyarrhythmia and hyperkalaemia may be from other co-morbidities.

Digoxin reduces atherosclerosis in apolipoprotein E-deficient mice.

BACKGROUND AND PURPOSE: Numerous in vitro studies have suggested that digoxin suppresses inflammation and alters lipid metabolism. However, the effect of dioxin on atherosclerosis is poorly understood. The present study was conducted to determine whether digoxin affects the development of atherosclerosis in a murine model of atherosclerotic disease. EXPERIMENTAL APPROACH: Apolipoprotein E-deficient mice maintained on a Western-type diet were administered PBS (control), low-dose digoxin (1 mg · kg(-1) · day(-1)) or high-dose digoxin (2 mg · kg(-1) · day(-1)) via i.p. injection for 12 weeks. KEY RESULTS: Digoxin dose-dependently reduced atherosclerotic lesion formation and plasma lipid levels (reductions of 41% in total cholesterol, 54% in triglycerides and 20% in low-density lipoprotein cholesterol in the high-dose digoxin-treated group). Moreover, treatment with digoxin markedly attenuated IL-17A expression and IL-17A-related inflammatory responses and increased the abundance of regulatory T cells (Tregs). CONCLUSIONS AND IMPLICATIONS: Our data demonstrate that digoxin acts as a specific antagonist of retinoid-related orphan receptor-γ to decrease atherosclerosis by suppressing lipid levels and IL-17A-related inflammatory responses.

Effect of Chinese Medicine Danshen and Indian Ayurvedic Medicine Bark of Arjuna Tree on a Relatively New LOCI Digoxin Assay for Application on the Vista 1500 Analyzer.

BACKGROUND: Danshen is a traditional Chinese medicine and bark of Arjuna tree is an Ayurvedic medicine both indicated as heart tonic. Interference of Danshen in serum digoxin immunoassays has been reported but potential interference of extract of bark of Arjuna tree has not been reported. We studied potential interferences of Danshen and bark of Arjuna tree on a relatively new LOCI digoxin assay for application on the Vista 1500 analyzer (Siemens Diagnostics). METHODS: Aliquots of drug-free serum were supplemented with ethyl acetate extract of Danshen (two different brands studied) or aqueous or ethyl alcohol extract of bark of Arjuna tree and apparent digoxin concentrations were measured by the LOCI digoxin assay. In another experiment, aliquots of serum pool containing digoxin were further supplemented with Danshen or bark of Arjuna tree extract and digoxin concentrations were measured again using LOCI digoxin assay. RESULTS: Little apparent digoxin concentration was observed when aliquots of drug-free serum pools were supplemented with Danshen or bark of Arjuna tree extract. When aliquots of serum digoxin pool were further supplemented with these extract, we observed statistically significant negative interference but such differences may not be clinically significant. CONCLUSION: We conclude that LOCI digoxin assay is virtually free from interferences of Danshen and extract of bark of Arjuna tree.

Hypoglycemia associated with oleander toxicity in a dog.

Oleander poisoning typically results in cardiac arrhythmias, hyperkalemia, and gastrointestinal irritation, and can be fatal. Oleander extracts have also been studied experimentally as hypoglycemic agents. Here, we describe a dog with confirmed oleander toxicosis presenting with classical symptoms and also hypoglycemia. After excluding other likely causes of hypoglycemia, the finding was attributed to oleander toxicosis, which has not been previously reported in dogs. A 7-year-old female spayed Maltese was presented to the emergency service after ingesting oleander leaves. Toxicosis was confirmed by measurement of digoxin using a competitive binding immunoassay, patient level 0.7 ng/mL (0.9 nmol/L) 24-h post-ingestion. Clinical symptoms included vomiting, cardiac arrhythmia, mild hyperkalemia, and hypoglycemia. Treatment was successful with aggressive supportive care, and the dog was discharged from the hospital after 48 h and made a full recovery. This case reviews the presentation and treatment of oleander toxicity but also highlights possible effects of oleander on blood sugar in dogs. Hypoglycemia in this dog, attributed to oleander poisoning, is interesting as it supports experimental research into hypoglycemic properties of oleander extracts.

Pharmacokinetic compatibility of ginsenosides and Schisandra Lignans in Shengmai-san: from the perspective of p-glycoprotein.

BACKGROUND: Phytochemical-mediated alterations in P-glycoprotein (P-gp) activity may result in herb-drug interactions by altering drug pharmacokinetics. Shengmai-san, a traditional Chinese herbal medicine composed by Panax Ginseng, Ophiopogon Japonicus, and Schisandra Chinensis, is routinely being used for treating various coronary heart diseases. In our previous studies, Schisandra Lignans Extract (SLE) was proved as a strong P-gp inhibitor, and herein, the compatibility of Shengmai-san was studied by investigating the influence of SLE on the pharmacokinetics of the ginsenosides from the perspective of P-gp. METHODOLOGY: Pharmacokinetic experiments were firstly performed based on in vitro uptake, efflux and transport experiments in Caco-2, LLC-PK1 wild-type and MDR1-overexpressing L-MDR1 cells. During the whole experiment, digoxin, a classical P-gp substrate, was used as a positive control drug to verify the cells used are the valid models. Meanwhile, the effects of SLE on the pharmacokinetics of ginsenosides were further investigated in rats after single-dose and multi-dose of SLE. RESULTS AND CONCLUSIONS: The efflux ratios of ginsenoside Rb2, Rc, Rg2, Rg3, Rd and Rb1 were found more than 3.5 in L-MDR1 cells and can be decreased significantly by verapamil (a classical P-gp inhibitor). Contrarily, the efflux ratios of other ginsenosides (Rh1, F1, Re, and Rg1) were lower than 2.0 and not affected by verapamil. Then, the effects of SLE on the uptake and transport of ginsenosides were investigated, and SLE was found can significantly enhance the uptake and inhibit the efflux ratio of ginsenoside Rb2, Rc, Rg2, Rg3, Rd and Rb1 in Caco-2 and L-MDR1 cells. Besides, In vivo experiments showed that single-dose and multi-dose of SLE at 500 mg/kg could increase the area under the plasma concentration time curve of Rb2, Rc and Rd significantly without affecting terminal elimination half-time. In conclusion, SLE could enhance the exposure of ginsenosides Rb2, Rc, Rg2, Rg3, Rd and Rb1 significantly.

Development of a dynamic multiple reaction monitoring method for determination of digoxin and six active components of Ginkgo biloba leaf extract in rat plasma.

A new liquid chromatography-tandem mass spectrometry (LC-MS/MS) method by using dynamic multiple reaction monitoring (DMRM) has been developed and validated for the simultaneous determination of digoxin (DGX) and six main components of Ginkgo biloba leaf extract (GBE) in rat plasma. Comparing with the conventional multiple reaction monitoring (MRM), DMRM dramatically decreases the number of concurrent MRM transitions, and significantly extended the dwell time, which provided much higher sensitivity and reproducibility than MRM when complex multi-component samples were quantified. The plasma samples were protein precipitated with methanol, the detection was accomplished with electro-spray ionization (ESI) as the ion source operating in the negative ionization mode, with methanol and water as mobile phase, and with an Agilent Zorbax eclipse plus C18 column (4.6 × 100 mm, 3.5 µm) as the analytical column. The total run time was 12.0 min. The validation of the method was implemented including specificity, linearity, accuracy, precision, recovery, matrix effect and stability. This method was successfully applied to the herb-drug pharmacokinetic interaction study of DGX combined with GBE after oral administration to rats. The result indicated that co-administration of GBE and DGX significantly influenced the pharmacokinetics of DGX when compared to that of single DGX-treated rats.

Down-regulation of P-gp expression and function after Mulberroside A treatment: potential role of protein kinase C and NF-kappa B.

P-Glycoprotein (P-gp) plays a major role in drug-drug and herb-drug interactions. Mulberroside A (Mul A) is one of the main bioactive constituents of Sangbaipi, the dried root-bark of Morus alba L. (white mulberry), which is officially listed in the Chinese Pharmacopoeia. In the present study, we investigated the effect of Mul A treatment on mRNA expression and protein expression of P-gp in the Caco-2 cells by real-time qPCR and Western blot analysis. The effect of Mul A treatment on the function of P-gp in vitro and in vivo was assessed by Rho123 transport assay and a pharmacokinetic study. The potential roles of protein kinase C (PKC) and nuclear factor kappa B (NF-κB) in the expression regulation of P-gp after Mul A treatment were also investigated. The results revealed that Mul A treatment significantly decreased the mRNA and protein expression of P-gp in Caco-2 cells after treatment with Mul A (5-20 µM). Furthermore, Mul A treatment displayed apparently inhibitory effect on the function of P-gp both in vitro and in vivo. In addition, activation of PKC activity and NF-κB nuclear translocation were observed in the presence of Mul A, which suggested that PKC and NF-κB might play crucial roles in Mul A-induced suppression of P-gp. Our study demonstrated that Mul A treatment could down-regulate P-gp expression and function accompanied by the activation of PKC and NF-κB, and this should be taken into consideration in potential herb-drug interactions when Mul A or M. alba are co-administered with other drugs transported by P-gp.

Bidirectional (negative/positive) interference of oleandrin and oleander extract on a relatively new Loci digoxin assay using Vista 1500 analyzer.

BACKGROUND: Oleander interferes with serum digoxin measurements using various immunoassays. The potential interference of oleander and its active ingredient, oleandrin, with a relatively new homogenous sequential chemiluminescent digoxin assay based on luminescent oxygen channeling technology (LOCI digoxin assay, Siemens Diagnostics) has not been previously reported. METHODS: Aliquots of a digoxin-free serum pool were supplemented with increasing concentrations of oleandrin, or with oleander extract, followed by measuring the apparent digoxin concentrations using the LOCI digoxin assay using Vista 1500 analyzer. Mice were fed oleandrin or oleander extract, and their blood digoxin levels at 1 and 2 h were measured with the LOCI digoxin assay. In addition, two digoxin serum pools were prepared by combining sera of patients receiving digoxin; aliquots of both pools were supplemented with oleandrin or oleander extract and digoxin concentrations were again measured. Attempts to overcome this interference were made by measuring free digoxin concentration using a third digoxin pool. RESULTS: Significant apparent digoxin concentrations were observed after supplementing aliquots of the drug-free serum pool with oleandrin or oleander extract. Mice fed with oleandrin or oleander extract also showed apparent digoxin levels 1 and 2 h after feeding. Digoxin values were also falsely lower or elevated (bidirectional interference) when aliquots of digoxin serum pools were further supplemented with oleandrin or oleander extract depending on concentration; this interference was not eliminated by free digoxin monitoring. CONCLUSIONS: Oleandrin interferes with LOCI digoxin assay.

The new LOCI digoxin assay on the Vista 1500 analyzer is virtually free from interferences of herbal supplements hawthorn and ashwagandha (Indian ginseng).

Herbal supplements hawthorn and ashwagandha (Indian ginseng) are indicated for cardiac illnesses and may be taken by patients receiving digoxin therapy. Because both hawthorn and ashwagandha are known to interfere with serum digoxin measurements using certain digoxin immunoassays, we investigated potential interference of these two herbal supplements with the new homogenous sequential chemiluminescent assay for digoxin based on the luminescent oxygen channeling technology (LOCI digoxin) for application on the Dimension and Vista platform. When aliquots of a drug-free serum pool were supplemented with various amounts of hawthorn (three different commercial preparations) or ashwagandha (two different commercial preparations) and apparent digoxin values were measured using LOCI digoxin assay on Dimension Vista 1500 analyzer we observed none-detected values except when aliquots were supplemented with very high amounts of the herbal extracts. When aliquots of a serum digoxin pool (prepared by pooling specimens from patients receiving digoxin) where further supplemented with various amounts of these supplements and digoxin concentrations were remeasured, statistically significant falsely higher digoxin values were observed only in specimens containing very high amounts of these supplements. Such interference may not be clinically significant. We conclude that new LOCI digoxin assay is virtually free from interferences of herbal supplements, hawthorn, and ashwagandha.

Clinically insignificant negative interferences of spironolactone, potassium canrenoate, and their common metabolite canrenone in new dimension vista LOCI digoxin immunoassay.

Spironolactone, a potassium-sparing diuretic metabolized to canrenone is often used with digoxin to treat various conditions including congestive heart failure. Potassium canrenoate is a similar drug, which is also metabolized to canrenone. Due to reported both positive and negative interference of spironolactone, potassium canrenoate, and their common metabolite canrenone with digoxin immunoassays, we investigated potential interference of these compounds with the new homogenous sequential chemiluminescent assay for digoxin based on the luminescent oxygen channeling technology (LOCI digoxin) for application on the Dimension and Vista platform. When aliquots of a drug-free serum pool were supplemented with various amounts of spironolactone, potassium canrenoate, or canrenone and apparent digoxin values were measured using Dimension Vista LOCI digoxin assay, we observed no detected value except when aliquots were supplemented with very high amounts of potassium canrenoate or canrenone. However, we observed that apparent digoxin concentrations were very low. When aliquots of a serum digoxin pool (prepared by pooling specimens from patients receiving digoxin), were further supplemented with various amounts of spironolactone, potassium canrenoate, or canrenone and serum digoxin concentrations were remeasured using the LOCIdigoxin assay, only statistically significant falsely lower digoxin values (negative interference) were observed in specimens containing very high amounts of canrenone or potassium canrenoate. However, such small bias may not have any clinical significance. We conclude that new Dimension Vista LOCI digoxin assay is virtually free from interferences of spironolactone, potassium canrenoate, and their common metabolite canrenone.

Analysis of saikosaponins in rat plasma by anionic adducts-based liquid chromatography tandem mass spectrometry method.

Saikosaponins (SSs) are a class of triterpene saponins with a wide spectrum of bioactivities. A sensitive liquid chromatography tandem mass spectrometry (LC-MS/MS) method was developed for simultaneous determination of saikosaponin a, saikosaponin c, saikosaponin d and saikosaponin b2 in rat plasma. Plasma samples were prepared by liquid-liquid extraction. The analytes and the internal standard (IS) digoxin were well separated on an octadecyl column using gradient elution and analyzed by monitoring the fragmentation transition pair of anionic adducts to deprotonated molecules in negative-mode electrospray. By neutral loss of HCOOH, the transition pairs of m/z 825 → 779 for SSa, SSd, SSb2 and the IS, and m/z 971 → 925 for SSc were sensitive for MS/MS detection with the lower limits of quantification in the range of 0.20-0.40 ng/mL. Method validation experiments were performed, including selectivity, precision, accuracy, linearity, matrix effect, recovery and stability. The validated method was further applied to determine the pharmacokinetics parameters of SSa, c and d in rats following a single oral administration of the extract of chaihu (the dried roots of Bupleurum chinense DC).

Unexpected double lethal oleander poisoning.

Nerium oleander is a very popular urban ornamental plant in Europe, but it is also extremely dangerous because it contains several types of glycosides, accidental ingestion of which can cause cardiac arrhythmias and even deaths. The rarity of such cases makes it difficult to think of oleander poisoning without evidences that suggest this possibility as the cause of the unexpected death. This report concerns the discovery of the bodies of 2 young people, a man and a woman, in a forest in conditions of extreme malnutrition. Medicolegal investigations showed neither pathologic nor traumatic causes of death, but the presence of vegetal remains in the stomach was noticed. A common toxicological analysis resulted negative, but the implementation of more detailed investigations showed the presence of digoxin in the blood of both cadavers, excluding the possibility of a pharmaceutical provenience of digoxin, this laboratory result was interpreted as evidence of ingestion of oleander, which contains oleandrine, the cross reaction of which with digoxin is widely described in the literature. Identification of the 2 subjects, which occurred after 4 years, strengthened the hypothesis of accidental poisoning by oleander because it was ascertained that the 2 young people were vegans--extreme vegetarians who reject the ingestion of foods of animal origin and live by eating only what they find in nature.