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Aconitum alkaloid poisoning is a type of poisoning caused by accidental ingestion and clinical use of herbal drugs in many countries. In this study, we developed an in-syringe dispersive micro solid-phase extraction (DMSPE) method for selective extraction of aconitine, benzoylaconine and aconine from human urine using a type of polymer material. All of the parameters influencing the extraction efficiency such as the type and amount of sorbent, the extraction time and the desorption solvent and volume in DMSPE were carefully investigated and optimized. Using DMSPE method, the absence of evaporation and centrifugation steps reduced the consuming time of sample preparation. Samples were analyzed by ultra high-performance liquid chromatography-high-resolution mass spectrometry on an HSS T3 analytical column. The results showed that the DMSPE method yielded fewer relative and absolute matrix effects, which reduced the sample to sample variability in human urine. The limits of detection and limits of quantitation of this method were determined to be 0.08-0.1 and 0.2-0.3 μg/L, respectively. The average recoveries of the analytes were between 88.6% and 107.2% with the intra- and interday precisions ranging from 2.1% to 6.4% and from 5.9% to 13.9%, respectively. The method presented here is an efficient, low-cost and selective extraction of aconitine, benzoylaconine and aconine from human urine., (© The Author 2017. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oup.com.)

Lappaconitine (LAP), a non-addictive potent analgesic drug, is broadly used to treat cancer and postoperative pain in many countries, and it also has antibiotic activity against Pseudomonas aeruginosa and Salmonella Typhi. Despite its widespread usage and potential for expanded use, its metabolism was poorly investigated. In this work, the metabolic fate of LAP in liver microsomes of the rat and human was compared, and after oral administration, the metabolites in the rat were investigated using ultra high-performance liquid chromatography-quadrupole time-of-flight mass spectrometry (UHPLC-Q/TOF-MS). As a result, a total of 51 metabolites were identified, including 48 metabolites that were reported here for the first time. Based on accurate MS/MS spectra and the known structure of LAP, the metabolites structures and their fragment ions were readily characterized. The biotransformations of LAP in vitro and in vivo were shown to involve hydroxylation, N-deacetylation, O-demethylation, N-deethylation, and hydrolysis. Furthermore, the results indicated a quantitative species difference in the metabolites for LAP between the rat and human. However, 16-DMLAP, DAL and 5'-OH-DAL were the main in vitro and in vivo metabolites. This work provides the LAP metabolite profiles in rat and human, which will help better understand the pharmacological and toxicological activities of LAP., (Copyright © 2015 Elsevier B.V. All rights reserved.)

The diester-diterpene alkaloid aconitine was quantified by liquid chromatography-tandem mass spectrometry in post-mortem specimens of three cases where suicidal ingestion of Aconitum napellus L. ('monkshood') was supposed. In an attempt at rationalization, sample preparation and chromatographic conditions of plasma/serum drug analysis routine were utilized. Linearity was established from 0.5 to 20 µg L⁻¹ using newborn calf serum (NCS) as a surrogate calibration matrix for all sample types and mesaconitine as an internal standard. Validation (selectivity, sensitivity, precision, accuracy, recovery of the extraction procedure, matrix effect, processed sample stability) confirmed the applicability of the analytical method to various post-mortem matrices. Internal standard selection was based on multi-matrix process efficiency data. In human post-mortem peripheral blood a lower limit of quantification of 0.51 µg L⁻¹ and a limit of detection of 0.13 µg L⁻¹ were accomplished (0.1 ml sample aliquots). Aconitine was degraded to a large extent in different sample types when being stored at +20 °C for 30 days, while at -20 °C and for some matrices also at +4 °C no appreciable degradation occurred. Aconitine concentrations in real samples were 10.3-17.9 µg L⁻¹ (peripheral blood, n = 3), 14.9-87.9 µg L⁻¹ (heart blood, n = 3), 317-481 µg L⁻¹ (urine, n = 2), 609-4040 µg L⁻¹ (stomach content, n = 3), 139-240 µg L⁻¹ (bile, n = 2), 8.4 µg L⁻¹ (vitreous humor, n = 1), 54.7 µg L⁻¹ (pericardial fluid, n = 1), 492 µg kg⁻¹ (liver, n = 1), 15.2-19.7 mg L⁻¹ (unknown liquids secured onsite, n = 3). Together with concomitant circumstances the analytical data provided compelling evidence for acute Aconitum poisoning as being the cause of death., (Copyright © 2013 John Wiley & Sons, Ltd.)

A rapid, specific and sensitive ultra performance liquid chromatography-mass spectrometry (UPLC-MS/MS) method was developed for the simultaneous determination of four trace alkaloids, including aconitine (AC), hypaconitine (HA), mesaconitine (MA) and yunaconitine (YA) in human urine samples. UPLC-MS-MS system coupled with an electrospray ionization (ESI) source was performed in multiple reaction monitoring (MRM) mode. The sample preparation was performed with hollow fiber microextraction (HF-LPME) prior to the analysis. The enrichment factors of the four alkaloids were 102 - 301. The separation was applied on a Waters ACQUITY UPLC BEH C18 column (50 mm x 2.1 mm, 1.7 micro m) with a gradient elution of acetonitrile and 10 mmol/L NH4HCO3, as mobile phase. The retention times , were less than 3 min. This method significantly improved the detection sensitivity, and the limits of quantitation were from 0. 01 to 0.1 ng/L. The calibration curves were linear over the ranges of 0.01 - 10 ng/L for AC, MA and YA, 0.1 - 100 ng/L for HA in human urine samples, and the correlation coefficients were 0. 998 1, 0.998 4, 0.999 5 and 0.998 6, respectively. The method was proved to be rapid and sensitive for aconitum alkaloid analysis in urine samples.

A sensitive and rapid high-performance liquid chromatographic-tandem mass spectrometric (HPLC-MS/MS) method was developed for the quantitation of the major metabolite of aconitine, 16-O-demethylaconitine, lappaconitine as the internal standard in rat urine. Urine samples were precipitated with acetonitrile/methanol (3:1, v/v). Chromatographic separation was achieved on a Kromasil C18 analytical column. Detection was performed by a selective reaction monitoring (SRM) mode via an electrospray ionization (ESI) source operating in the positive ionization mode. The analytical method was validated in terms of specificity, precision, accuracy, extraction recovery. The intra- and inter-day precisions were less than 11.7%, and the accuracy was less than 14.0% for the analyte. The validated method has been applied to a pharmacokinetic study of 16-O-demethylaconitine in rats, following oral administration of aconitine.

Hollow fiber liquid-phase microextraction (HF-LPME) coupled with high-performance liquid chromatography was used to simultaneously determine three Aconitum alkaloids, including aconitine (AC), hypaconitine (HA) and mesaconitine (MA) in human urine sample. Analytes were extracted from 5mL urine sample containing 1.0mmol/L NaOH into 1-octanol membrane phase impregnated in the pores of hollow fiber wall, and then back extracted into acidified aqueous solution in the lumen of the hollow fiber. After extraction, 10μL of the acceptor phase was analyzed directly by HPLC. In this method, some important extraction parameters, such as organic solvent, extraction time, stirring rate, pH of donor phase and acceptor phase, temperature, and the volume of acceptor phase were optimized. This method provided 98- to 288-fold enrichment factors within 60min of extraction and good repeatability with RSDs of 0.99-7.22%. The calibration curves were linear over the ranges of 16.0-128.0μg/L for AC, 11.0-88.0μg/L for HA and 8.1-64.8μg/L for MA in human urine sample, with correlation coefficients of 0.9949, 0.9969 and 0.9904, respectively. Limits of detection were from 0.7 to 1.5μg/L, and recoveries from spiked urine sample varied from 84.4% to 106.2% for AC, 77.3% to 85.6% for HA and 90.1% to 100.8% for MA., (Copyright © 2010 Elsevier B.V. All rights reserved.)

The mesaconitine and its major metabolites in the rat urine were identified by liquid chromatography and electrospray ionization tandem mass spectrometry. The rat urine was collected for consecutive 24 hours from the rat following intragastric infusion of mesaconitine, subsequently which were enriched and purified using solid phase extraction. The metabolites of mesaconitine in the rat urine were analyzed by the liquid chromatography and electrospray ionization tandem mass spectrometry. It is shown that the parent drug mesaconitine and its metabolites were found in the rat urine, such as hypo-mesaconitine glucuronic acid conjugate, 10-hydroxy-mesaconitine, 1-O-demethyl mesaconitine, deoxy-mesaconitine and hypo-mesaconitine. Among the five of metabolites, the hypo-mesaconitine glucuronic acid conjugate (m/z 766) was first discovered as the aconitine in rats phase II metabolites, which revealed a new way of mesaconitine metabolism in rats.

Objective: To investigate the distribution of aconite alkaloids in biological fluids and tissues in the corpse died of acute aconite intoxication and to provide information for sample selection and result evaluation in forensic identification., Methods: The content of aconite alkaloids in biological fluids and tissues were determined by liquid chromatography-tandem mass spectrometry., Results: The content of aconite displayed in decending order of urine, bile, gastric content, heart blood, pancreas, heart, intestine, liver, kidney, stomach, lung, gallbladder and spleen, with no aconite detected in the brain., Conclusion: It was indicated that urine, bile and blood are the best specimens for the determination of aconite in body of the acute aconite intoxication.

Aconite poisoning was examined in five patients (four males and one female) aged 49 to 78 years old. The electrocardiogram findings were as follows: ventricular tachycardia and ventricular fibrillation in case 1, premature ventricular contraction and accelerated idioventricular rhythm in case 2, AIVR in case 3, and nonsustained ventricular tachycardia in cases 4 and 5. The patient in case 1 was given percutaneous cardiopulmonary support because of unstable hemodynamics, whereas the other patients were treated with fluid replacement and antiarrhythmic agents. The main aconitine alkaloid in each patient had a half-life that ranged from 5.8 to 15.4 h over the five cases, and other detected alkaloids had half-lives similar to the half-life of the main alkaloid in each case. The half-life of the main alkaloid in case 1 was about twice as long as the half-lives in the other cases, and high values for the area under the blood concentration-time curve and the mean residence time were only observed in case 1. These results suggest that alkaloid toxicokinetics parameters may reflect the severity of toxic symptoms in aconite poisoning.

Poisoning from aconite occurs worldwide as a result of misuse of the potent plant. Laboratory investigation into suspected intoxication cases is challenging because the content of toxic aconitum alkaloids varies depending on the plant source, market processing, dosing protocol, hydrolytic degradation, and metabolic transformation. Using a triple-quadrupole tandem mass spectrometer, a group screening method was developed based on the mass-fragmentographic scheme of common aconitum alkaloids. The precursor-ion scans of m/z 105 and 135 permitted selective profiling of 14-O-benzoyl-norditerpenoids and the 14-O-anisoyl-norditerpenoids, respectively. Gradient reversed-phase liquid chromatography minimized coelution of isobaric compounds. The screening protocol was applied to a clinical investigation of suspected herbal poisoning. In total, 15 urine samples were thus screened positive for aconitum alkaloid over 5 years. The diagnoses of aconite poisoning in 11 patients were firmly established based on the known prescription history and the positive urine finding. In four patients, without aconitum herbs being listed in the herbal prescriptions, contamination of the herbal remedies by aconite was suspected to be the hidden cause of their acute poisoning. Yunaconitne, a highly toxic aconitum alkaloid, was thus identified in human urine for the first time. The group screening method of aconitum alkaloids in urine is an important diagnostic aid for acute poisoning by aconites of an unclear origin.

An HPLC method has been developed for the simultaneous determination of the toxic Aconitum alkaloids, aconitine, mesaconitine and hypaconitine in blood and urine samples. The samples were initially subjected to solid phase extraction using Oasis MCX cartridges, and the alkaloids were separated on an XTerra RP18 column, gradient-eluted with acetonitrile: ammonium hydrogen carbonate buffer. Calibration curves were linear in the range 2.75-550 ng for aconitine and hypaconitine, and 3-600 ng for mesaconitine: the limit of detection was 0.1 ng (signal-to-noise ratio of 3) for each alkaloid. The described analysis proved to be sensitive, rapid and economical, and will be applied in the identification and determination of these alkaloids in forensic and therapeutic drug monitoring.

Aim: To identify the main metabolites of aconitine in the urine of rabbits., Methods: After oral administration of aconitine (5 mg.kg-1), the urine of male rabbits was collected and extracted by solid phase extraction and analyzed by liquid chromatography-ion trap mass spectrometry., Results: Aconitine and 4 metabolites were found in the rabbit urine. Their protonated molecular ions at m/z 632, m/z 604, m/z 590, m/z 500 and multistage fragment ions with neutral loss of 60 u, 32 u, 28 u and 18 u were monitored. Their relative concentration were M1 > Aconitine > M4 > M2 > M3., Conclusion: The metabolites M1-M4 were deduced as 16-O-demethylaconitine, benzoylaconine, 16-O-demethylbenzoylaconine and aconine, respectively.

Determination of fourteen alkaloids, toxic Aconitum alkaloids, aconitine, mesaconitine, jesaconitine, hypaconitine and deoxyaconitine, and their hydrolysis products, benzoylaconines and aconines, have been established using capillary liquid chromatography (LC) fast atom bombardment mass spectrometry (FAB-MS) with a frit interface. Protonated molecular ions were observed as base peaks in the FAB-MS for these fourteen alkaloids. All the alkaloids were simultaneously quantified with linear gradient LC elution by solvent mixture of acetonitrile and 0.3% trifluoroacetic acid using selected ion monitoring of the protonated molecular ions. The calibration curves of these alkaloids were linear in injection amounts ranging from 5 to 500 pg, and their detection limits were 1 pg per injection (S/N=3). Solid-phase extraction using Sep-Pak Plus PS-1 was also investigated to clean-up and concentrate alkaloids in blood and urine samples, and showed satisfactory recoveries. This capillary LC-frit-FAB-MS method enables determination of low levels of Aconitum alkaloids in blood and urine samples, coupled with solid-phase extraction.

Described here is a fatal case of accidental aconitine poisoning following the ingestion of aconite, Torikabuto, mistaken for an edible grass, Momijigasa. A 61-year-old man developed symptoms of nausea, diarrhea, and discomfort of the body about 2 h after the ingestion and was taken to an emergency room. Resuscitation and antiarrhythmic drugs were ineffective, and ventricular tachycardia and fibrillation developed and lasted for 6 h. He was transferred to a coronary care unit and complete sinus rhythm was obtained on an electrocardiogram 30 h after his admission. The patient fell into a coma and died of brain edema diagnosed by CT on the 6th day. Consent for autopsy was denied by the family but was given for gas chromatography/selected ion monitoring (GC/SIM) to analyze the toxicity of aconitine alkaloids in the blood and the urine. Only a faint amount of jesaconitine was detected, while aconitine, mesaconitine and hypaconitine were not detectable in the blood 24 h after ingestion. On the other hand, aconitine and its related alkaloids such as mesaconitine, jesaconitine, and hypaconitine were clearly detected in the urine.

The separation and characterization of the metabolic products of lappaconitine in rat urine by high-performance liquid chromatography with electrochemical and ultraviolet detection are described. Urine samples from rats intravenously administered lappaconitine hydrobromide were extracted with chloroform and then purified on a Sep-Pak C18 cartridge. The subsequent resolution into individual compounds was achieved by high-performance liquid chromatography. Identification of these compounds was based on comparisons of the chromatographic behaviour and the detector response with those of authentic samples. Changes in the ratio of lappaconitine to its metabolites in rat urine with time after dosing led to a proposal for one of the probable metabolic pathways of lappaconitine in the rat.

The metabolites of lappaconitine in the urine of humans having been previously administered intramuscularly with lappaconitine hydrobromide were studied using high performance liquid chromatography with electrochemical and ultraviolet detection. The urine was extracted by means of liquid- and solid-phase extractions. Each of the metabolites of lappaconitine was purified by high performance liquid chromatography on a reversed phase column and identified on the basis of the chromatographic behaviour and the detector response. It was proved that lappaconitine, N-deacetyl-16-O-demethyllappaconitine and N-deacetyllappaconitine were excreted in urine from humans receiving lappaconitine.

A sensitive method is presented for determining aconitine. Aconitine is complexed with Co2+, the aconitine-cobalt complex is extracted with chloroform, and the absorbance is measured at 320 nm. The sensitivity of the method ranged between 0.06 and 3 mg/25 ml, and the color was stable for 6 hr. The method was successfully applied for the quantitative determination of aconitine in animal tissues.