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White analytical chemistry is a novel concept for the assessment of analytical methods on basis of its validation efficiency, greenness power, and economical efficiency. White analytical chemistry-driven stability indicating chromatographic method has been developed for the concomitant analysis of thiocolchicoside and aceclofenac. The proposed chromatographic method has been developed using a safe and environmental-friendly organic solvents for the concomitant stability study of thiocolchicoside and aceclofenac. The analytical risk assessment was carried out for the identification of high-risk analytical risk factors and analytical method performance attributes. The mixture design was applied for the design of experiments-based response surface modeling of high-risk analytical risk factors and analytical method performance attributes. The degradation products were isolated and characterized using infrared, nuclear magnetic resonance, and mass spectral data. The proposed method was compared for its validation efficiency, greenness power, and cost-efficiency with published chromatographic methods using the red, green, and blue models. The white score of the proposed and reported method was calculated by averaging the red, green, and blue scores of the methods. The proposed method was found to be robust, green, and economical for the concomitant stability study of thiocolchicoside and aceclofenac., (© 2023 Wiley-VCH GmbH.)

Colchicine (COL) has been used to treat gout for over a millennium, but its medicinal use has been controversial due to its potent toxicity in the gastrointestinal tract. Nausea, vomiting, and diarrhea are the most prominent external manifestations of COL gastrointestinal toxicity, but the cause of these adverse events remains obscure. In this study, the mice were exposed to COL (2.5 mg/kg b.w./day) for one week to study the mechanism of COL-induced diarrhea from the perspective of intestinal metabolism. The results showed that COL exposure disturbed intestinal metabolic homeostasis, resulting in a significant accumulation of 116 metabolites and, conversely, significant depletion of 64 metabolites, with the number of differential metabolites being one-eighth of the total metabolites (180/1445). Also, it was found that cAMP, Adenosine 5'-monophosphate, GDP, Inositol, and Cortisol are core metabolites that play crucial roles in COL-induced metabolic disorders. These metabolites could be used as biomarkers to differentiate control and COL-treated groups, implying that these metabolites may be closely related to COL-induced diarrhea. Furthermore, changes in the metabolic pathways (Purine metabolism, biosynthesis and metabolism of aromatic amino acids, and Bile secretion) involved in these five core metabolites increased the toxic load in the gut, which was the culprit leading to intestinal metabolic disorders. In addition, the abnormal bile secretion caused by COL exposure may play an important role in COL-induced diarrhea. In conclusion, our study opens new avenues for understanding the mechanisms of COL-induced gastrointestinal adverse reactions and broadens the scientific horizon on the interactions between COL and host metabolism., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2022 Elsevier B.V. All rights reserved.)

The variation in alkaloid metabolites (colchicine and gloriosine) was found significant in the nine germplasms of G. superba (L.), collected from Central India. The maximum content of colchicine and gloriosine was in NBG-15 (Chitrakoot, M.P) and NBG-13 (Bheraghat, M.P). The phenolic acids viz . quercetin and kaempferol was first ever quantified in G. superba tuber. Cluster analysis on chemical variability (colchicine and gloriosine content) results in the identification of three elite germplasm(s). The radical scavenging potential was also found promising in the selected elite germplasm viz . NBG-13, NBG-14 and NBG-15. Further, the protein denaturation potential of elite chemotypes was found at par with standard colchicine. The study will aid in site specific exploration of high metabolite yielding chemotype(s) with validated pharmacological action to meet out the industrial demands. This will also promotes the commercial cultivation of species for socio economical upliftment in the area having similar phyto geographical conditions.

Gloriosa superba L. has economic significance due to colchicine, a bioactive compound used for gout. In present study metabolic and molecular variability in natural population of species was analyzed and correlated with edaphic and climatic factors. Thirty populations (wild) of G. superba were mapped from 10 different eco-regions of India at an elevation range of 10-1526 m, having no morphotypic variations. The two known biologically active alkaloids colchicine (ranged from 0.015-0.516%) and gloriosine (0.19-0.44%) were significantly varied (p < 0.05) among populations, leading to the identification of four elite chemotypes. Molecular variability from ISSR data divides the population in different sub clusters at intra-specific level, presenting the high similarity percentage with bootstrap value of 66-100%. Principal component analysis (PCA) revealed that elite chemotypes are related to temperature, precipitation and aridity gradient. The rhizospheric soil selenium was significantly correlated with colchicine content in G. superba., (Copyright © 2021 Elsevier B.V. All rights reserved.)

Background: Gloriosa superba L. (Colchicaceae) is a high-value medicinal plant indigenous to Africa and Southeast Asia. Its therapeutic benefits are well-established in traditional medicines including Ayurveda. It is well known for its natural bioactive compound colchicine which exhibits a wide range of pharmacological activities i.e. rheumatism, gout and was also introduced into clinical practices. The increasing demand as well as its illegal harvesting has brought this valuable plant under threatened category., Methods: The present investigation describes a microwave assisted extraction (MAE) strategy coupled with a densitometric-high performance thin layer chromatographic (HPTLC) methodology for the analysis of colchicine from 32 different populations of G. superba. A Box-Behnken statistical design (3 level factor) has been employed to optimize MAE, in which power of microwave, time of irradiation, aqueous ethanol and pH were used as independent variables whereas colchicine was used as the dependent variables. Chromatography was carried out on Silica gel 60 F 254 TLC plates with toluene: methanol, 85:15 (v/v) being used as solvent system. Densitometric measurement was performed at λ=254 nm following post-derivatization (10% methanolic sulphuric acid)., Results: Optimal conditions for extraction to obtain the maximum colchicine yield was found to be 7.51 mg g - 1 which was very close to be predicted response 7.48 mg g - 1 by maintaining microwave power (460 W), irradiation time (6.4 min), aqueous ethanol-30, pH -3. Colchicine content ranged between 2.12-7.58 mg g - 1 among 32 G. superba populations in which only three chemotypes viz. GS- 1, GS- 3, and GS- 2 collected from West Bengal and Sikkim, respectively exhibited maximum yield of colchicine., Conclusion: Therefore, this newly developed optimized MAE coupled with HPTLC densitometry methodology not only quantifies colchicine in order to identify elite chemotypes of G. superba, but it also encourages in selecting high yielding populations of the plants for industrial use and economic boost for the farmers. This validated, simple and reproducible HPTLC protocol is being used for the first time to estimate colchicine from natural populations of G. superba obtained from 32 different geographical regions of India.

Fungal endophytes, a major component of the plant host microbiome, are known to synthesize plant-derived metabolites in vitro. However, attenuation of metabolite production upon repeated sub-culturing is a major drawback towards utilizing them as an alternative for plant-derived metabolites. In this study, we isolated Diaporthe perseae, a fungal endophyte from Gloriosa superba tubers, which showed the production of colchicine in axenic cultures. Mass spectrometry, Nuclear Magnetic Resonance spectroscopy, and tubulin polymerization assays confirmed the compound to be colchicine. Repeated sub-culturing of the endophyte for 10 generations led to a reduction in the yield of the metabolite from 55.25 μg/g to 2.32 μg/g of mycelial dry weight. Treatment of attenuated cultures with DNA methylation inhibitor 5-azacytidine resulted in increased metabolite concentration (39.68 μg/g mycelial dry weight) in treated samples compared to control (2.61 μg/g mycelial dry weight) suggesting that 5-azacytidine can induce demethylation of the fungal genome to overcome the phenomenon of attenuation of metabolite synthesis. Reduced levels of global methylation were observed upon 5-azacytidine treatment in attenuated cultures (0.41 % of total cytosines methylated) as compared to untreated control (0.78 % of total cytosines methylated). The results provide a significant breakthrough in utilizing fungal endophytes as a veritable source of plant-derived metabolites from critically endangered plants., (Copyright © 2020 Elsevier B.V. All rights reserved.)

Background and Objective: Familial Mediterranean fever (FMF) is the most common monogenic autoinflammatory disease (AID) worldwide. The disease is caused by mutations in the MEFV gene encoding the inflammasome sensor Pyrin. Clinical diagnosis of FMF is complicated by overlap in symptoms with other diseases, and interpretation of genetic testing is confounded by the lack of a clear genotype-phenotype association for most of the 340 reported MEFV variants. In this study, the authors designed a functional assay and evaluated its potential in supporting FMF diagnosis., Methods: Peripheral blood mononuclear cells (PBMCs) were obtained from patients with Pyrin-associated autoinflammation with an FMF phenotype (n=43) or with autoinflammatory features not compatible with FMF (n=8), 10 asymptomatic carriers and 48 healthy donors. Sera were obtained from patients with distinct AIDs (n=10), and whole blood from a subset of patients and controls. The clinical, demographic, molecular genetic factors and other characteristics of the patient population were assessed for their impact on the diagnostic test read-out. Interleukin (IL)-1β and IL-18 levels were measured by Luminex assay., Results: The ex vivo colchicine assay may be performed on whole blood or PBMC. The functional assay robustly segregated patients with FMF from healthy controls and patients with related clinical disorders. The diagnostic test distinguished patients with classical FMF mutations (M694V, M694I, M680I, R761H) from patients with other MEFV mutations and variants (K695R, P369S, R202Q, E148Q) that are considered benign or of uncertain clinical significance., Conclusion: The ex vivo colchicine assay may support diagnosis of FMF and functional subtyping of Pyrin-associated autoinflammation., Competing Interests: Competing interests: None declared., (© Author(s) (or their employer(s)) 2020. Re-use permitted under CC BY. Published by BMJ.)

Aims: Vinegar-baked Radix Bupleuri (VBRB) potentiates the activity of anticancer drugs in the liver by increasing their hepatic distribution. However, this phenomenon may be associated with drug transporters. We investigated the effect of saikosaponin b2 (SSb2; the main component of VBRB) on the activity and expression of different drug transporters in both normal cells and those that overexpress the transporter., Main Methods: The activities of transporters were analyzed by concentration of their cellular substrates. Concentrations of colchicine (substrate of Pgp and MRP1) and cisplatin (substrate of OCT2 and MRP2) were determined by high-performance liquid chromatography (HPLC). The concentration of rhodamine B was determined by flow cytometry. The expression of transporter gene and protein were determined by qRT-PCR and Western blotting analysis., Key Findings: SSb2 increased colchicine efflux in HEK293 cells by primarily increasing Mrp1 activity, independent of gene and protein expression. SSb2 enhanced Mrp2 function and increased cisplatin efflux in BRL3A cells by upregulating Mrp2 gene expression, with a marginal effect on Pgp in normal cells. SSb2 increased OCT2 activity in OCT2-HEK293 cells by increasing the expression of OCT2 protein and mRNA; however, SSb2 inhibited MRP2 activity in MRP2-HEK293 cells by decreasing MRP2 protein expression, and decreased Pgp and MRP1 activity in Pgp- and MRP1-HEK293 cells., Significance: SSb2 might potentially be the key active component of VBRB that enhances the hepatotargeting of anticancer drugs through the inhibition of multidrug resistance-associated drug transporters (Pgp, MRP1, and MRP2) in an environment-dependent manner., (Copyright © 2019 Elsevier Inc. All rights reserved.)

Colchicine is highly toxic to creatures, and sensitively detecting colchicines is of profound significance in the pharmaceutical, clinical, food, and breeding industries. We herein designed and constructed a unique luminescent indium-organic framework {[(CH 3 ) 2 NH 2 ][In(L)]·9DMF} n ( V105 ) via an in situ ligand-mediated method, which possesses a 2-fold interpenetrated 3D anionic framework. Due to the large channels that exist in the framework with the size of 12 Å × 14 Å, V105 can rapidly remove harmful cationic dyes from water in a few minutes. Importantly, luminescent experiments demonstrate that V105 can selectively detect colchicine with high sensitivity in water, and the limit of detection can reach 1.0 × 10 -7 M. To our knowledge, this is the first example of a metal-organic framework-based luminescent sensor for detecting colchicine.

Rationale: A gouty tophus, arising from the deposition of monosodium urate crystals (MSU), rarely occurs in the nasal bridge. There have been only 7 documented cases of a gouty tophus in the nasal bridge from 1978 to 2018 in English-language literature., Patient Concerns: A 65-year-old male had a chief complaint of a lump in the nasal bridge that was slowly growing for over 1 year., Diagnosis: MSU crystals were confirmed through ultrasonography (US) and pathological examinations., Interventions: A cosmetically less destructive method, ultrasound-guided fine needle aspiration cytology (FNAC) was used to approach the mass lesion of nasal bridge., Outcomes: The diagnosis was confirmed as a gouty tophus without performing a nasal subdermal exploration., Lessons: This case report is the first use of US with FNAC to approach and diagnosed a gouty tophus in the nasal bridge.

In this study, a fluorescence polarization immunoassay (FPIA) technique was developed to determine colchicine (COL), an alkaloid of noxious plants of the order Liliales that is used in a number of medications to treat gout. An optimal combination of the polyclonal antibody and the antigen labelled with fluorescein isothiocyanate (FITC) was selected. Conditions for the competitive interaction of the antigen in the tested samples and its fluorophore conjugate (COL-FITC) with anti-COL antibodies were optimised, and the analytical characteristics of the assay were determined. The developed FPIA was characterised by a detection limit of 1.8 ng/mL and a detectable analyte concentration range of 4.1-74.3 ng/mL. The duration of the analysis was 10 min. The applicability of the developed FPIA for quality control of ready-made drug formulations and for the estimation of COL content in various matrices (urine, milk), with recovery values ranging from 79 to 108%, was demonstrated., (Copyright © 2018 Elsevier B.V. All rights reserved.)

A facile single-step preparation strategy for fabricating vancomycin functionalized organic polymer-based monolith within 100μm fused-silica capillary was developed. The synthetic chiral functional monomer, i.e 2-isocyanatoethyl methacrylate (ICNEML) derivative of vancomycin, was co-polymerized with the cross-linker ethylene dimethacrylate (EDMA) in the presence of methanol and dimethyl sulfoxide as the selected porogens. The co-polymerization conditions were systematically optimized in order to obtain satisfactory column performance. Adequate permeability, stability and column morphology were observed for the optimized poly(ICNEML-vancomycin-co-EDMA) monolith. A series of chiral drugs were evaluated on the monolith in either polar organic-phase or reversed-phase modes. After the optimization of separation conditions, baseline or partial enantioseparation were obtained for series of drugs including thalidomide, colchicine, carteolol, salbutamol, clenbuterol and several other β-blockers. The proposed single-step approach not only resulted in a vancomycin functionalized organic polymer-based monolith with acceptable performance, but also significantly simplified the preparation procedure by reducing time and labor., (Copyright © 2018 Elsevier B.V. All rights reserved.)

In present work, facile method is developed for determination of colchicine in human plasma sample, autumn and spring root of colchicium extracts by ultrasound assisted dispersive magnetic solid phase microextraction followed by HPLC-UV method (UAD-MSPME-HPLC-UV). Magnetic (Fe 2 O 4 -nanoparticles) metal organic framework-5, (MOF-5(Zn)-Fe 2 O 4 NPs) was synthesized by dispersing MOF-5 and Fe(NO 3 ) 3 .9H 2 O in ethylene glycol (as capping agent) and NaOH (pH adjustment agent) by hydrothermal method. The prepared sorbent was characterized via XRD and SEM analysis and applied as magnetic solid phase in UAD-MSPME-HPLC-UV method. In this method, colchicine molecules were sorbed on MOF-5(Zn)-Fe 2 O 4 NPs sorbent by various mechanisms like ion exchange, hydrogen bonding and electrostatic, ᴨ-ᴨ, hard-hard and dipole-ion interaction followed by exposing sonication waves as incremental mass transfer agent and then the sorbent was separated from the sample matrix by an external magnetic fields. Subsequently, accumulated colchicine were eluted by small volume of desorption organic solvent. Influence of operational variables such as MOF-5(Zn)-Fe 2 O 4 NPs mass, volume of extracting solvent and sonication time on response property (recovery) were studied and optimized by central composite design (CCD) combined with desirability function (DF) approach. Under optimum condition, the method has wide linear calibration rang (0.5-1700ngmL -1 ) with reasonable detection limit (0.13ngmL -1 ) and R 2 =0.9971. Finally, the UAD-MSPME-HPLC-UV method was successfully applied for determination of colchicine autumn and spring root of colchicium extracts and plasma samples., (Copyright © 2017 Elsevier B.V. All rights reserved.)

Clinical evaluation of a colchicine specific antigen-binding fragment (Fab) in order to treat colchicine poisoning required the development of an accurate method allowing quantification of free and Fab-bound colchicine in plasma and urine, and free colchicine in tissues, to measure colchicine redistribution after Fab administration. Three methods have been developed for this purpose, and validated in plasma, urine and liver: total colchicine was determined after denaturation of Fab by dilution in water and heating; free colchicine was separated from Fab-bound colchicine by filtration with 30KDa micro-filters; tissues were homogenized in a tissue mixer. Deuterated colchicine was used as internal standard. Samples were extracted by liquid-liquid extraction and analyzed with a LC-MS/MS. LOQ were 0.5ng/mL in plasma and urine for free and total colchicine and 5pg/mg in tissues. The methods were linear in the 0.5-100ng/mL range in plasma and urine, and 5-300pg/mg in tissues with determination coefficients>0.99. Precision and accuracy of QC samples presented a CV<9.4%. The methods require only 200μL of sample and allow a high throughput due to short analytical run (2min). These methods were successfully applied to a pig intoxicated with colchicine and treated with colchicine specific Fab fragments., (Copyright © 2017 Elsevier B.V. All rights reserved.)

Introduction: Horse feed material of Swiss and foreign production available on the Swiss market was tested with regard to possible contamination with 7 different alkaloids, atropine and colchicine. Twenty-eight feed samples as well as 2 poppy seed samples were analyzed. Out of 28 feed samples 18 were positive for prohibited substances in Equestrian sports. The concentration of prohibited substances was in none of the samples high enough to cause an effect on the body of the horse. The poppy seed sample, which was obtained by conventional harvesting and cleaning methods, contained a very high Morphine concentration compared to the sample which was harvested by hand. Despite high quality standards a contamination with prohibited substances in horse feed cannot be excluded.

A stability indicating high performance liquid chromatography method has been developed for the determination of thiocolchicoside (TCC) and its main degradation products thiocolchicoside S-oxide (D1SO) and 3-O-demethylthiocolchicine (D3) in liquid and solid formulations. The method was developed based on a previous forced degradation study showing that TCC underwent chemical degradation by acid/base catalyzed hydrolysis and oxidation being the main degradation products D3 and D1SO respectively. The analytes separation and quantification were achieved on a Synergi™ 4μm Polar-RP 80Å, column 150×4.6mm (Phenomenex) using the mobile phase constituted (flow rate 1mLmin -1 ) of eluant A: 20mM sodium acetate buffer (pH 5.0) and eluant B: MeOH:CH 3 CN (20:80); the elution was performed in gradient mode detecting the analytes at 254nm. The method showed linearity for TCC assay in the 5-15μgmL -1 , range and for unknown (TCC fu ) and known (D1SO and D3) degradation products assay, in the 0.5-10μgmL -1 range: all the square of the correlation coefficients were greater than 0.999. The precision, determined in terms of intra-day and inter-day were expressed as RSDs and resulted to be 1.19, 1.10, 1.37 and 1.04% and 0.95, 0.83, 1.30 and 0.72 for TCC, TCC fu , D1SO and D3, respectively. The method demonstrated also to be accurate; indeed, the average recoveries were 102.1/102.0% for TCC (ampoules and hard capsules respectively), 101.3/100.3% for TCC fu , 101.7/100.2% for D1SO, and 101.4/101.4% for D3. The robustness was also evaluated by variations of mobile phase composition and pH. Finally, the applicability of the method was evaluated by analysis of commercial liquid and solid dosage forms., (Copyright © 2016 Elsevier B.V. All rights reserved.)

Tubulin, which is the building block of microtubules, plays an important role in cell division. This critical role makes tubulin an attractive target for the development of chemotherapeutic drugs to treat cancer. Currently, there is no general binding assay for tubulin-drug interactions. The present work describes the application of the catch-and-release electrospray ionization mass spectrometry (CaR-ESI-MS) assay to investigate the binding of colchicinoid drugs to αβ-tubulin dimers extracted from porcine brain. Proof-of-concept experiments using positive (ligands with known affinities) and negative (non-binders) controls were performed to establish the reliability of the assay. The assay was then used to screen a library of seven colchicinoid analogues to test their binding to tubulin and to rank their affinities.

The semi-quantitative analysis of decomposed bone of rats exposed to colchicine and euthanized following different time intervals postexposure (i.e., dose-death interval, DDI) is described. Rats received colchicine (50 mg/kg, i.p.) and were euthanized 30 min (DDI1; n = 4), 60 min (DDI2; n = 4), or 180 min (DDI3; n = 4) postdose. Drug-free animals (n = 3) served as negative controls. Perimortem heart plasma was collected. Remains were decomposed to skeleton outdoors and then collected and sorted (skull, vertebrae, rib, pelvis, femur, tibia). Bones were dried, pulverized, and prepared by microwave-assisted extraction and microplate solid-phase extraction (MAE-MPSPE), followed by analysis for colchicine, 3-demethylcolchicine (3DMC), and 2-demethylcolchicine (2DMC) by ultra-high-performance liquid chromatography with photodiode array detection (UHPLC-PDA) at 350 nm. Bone type was a main effect (Kruskall-Wallis, p < 0.05) with respect to drug level (expressed as mass-normalized response ratio, RR/m) for each analyte, at each DDI. For all samples, DDI was a main effect (Kruskall-Wallis, p < 0.05) with respect to analyte level, and the ratio of analyte levels (RR3DMC/RRCOLCH, RR2DMC/RRCOLCH, and RR2DMC/RR3DMC). Bone COLCH levels varied by 19-fold, 12-fold, and 60-fold across all bone types in the DDI1, DDI2, and DDI3 groups, respectively. Bone 3DMC levels varied by 12-fold, 11-fold and 17-fold across all bone types in the DDI1, DDI2, and DDI3 groups, respectively. Bone 2DMC levels varied by 20-fold, 14-fold, and 14-fold across all bone types in the DDI1, DDI2, and DDI3 groups, respectively. Values of RR3DMC/RRCOLCH varied by 16-fold, 5-fold, and 5-fold across all bone types in the DDI1, DDI2, and DDI3 groups, respectively. Values of RR2DMC/RRCOLCH varied by 10-fold, 6-fold, and 12-fold across all bone types in the DDI1, DDI2, and DDI3 groups, respectively. Values of RR2DMC/RR3DMC varied by 3-fold, 5-fold, and 2-fold across all bone types in the DDI1, DDI2, and DDI3 groups, respectively. Measured analyte levels in bone correlated poorly with corresponding levels in blood (r = -0.65-+0.31). Measured values of RR2DMC/RRCOLCH and RR2DMC/RR3DMC in bone also correlated poorly with corresponding values in blood. Measured values of RR3DMC/RRCOLCH were well correlated with corresponding blood levels for all bone types except skull (r = 0.91-0.97).

A rapid, accurate, precise, robust and specific stability indicating RP-HPLC method has been developed and validated for simultaneous determination of fixed dose combination of Aceclofenac (ACF) and Thiocolchicoside (THC). Combinations and marketed tablets were subjected to stress conditions such as oxidation, hydrolysis, photolysis and heat. Successful separation of drugs from stress degradation products was achieved on Kromasil C18 (250 × 4.6 mm, 5 μm) column at 30 °C using gradient mobile phase system consisting of (A) 10 mM ammonium acetate pH 5.00 buffer and (B) acetonitrile: water (70:30 v/v). The flow rate was 1.0 mL/min with UV detection at 265 nm. The retention time of THC and ACF was 13.29 and 22.20 min respectively. Peak purity of both the drugs was passing in all degradation conditions demonstrates the specificity of assay method for their estimation in presence of degradation products. The developed HPLC method was validated for linearity, accuracy, precision and robustness. The linearity of the proposed method was investigated in the range of 80-280 µg/mL for ACF and 6.4-22.4 µg/mL for THC. The utility of the procedure was verified by its application to marketed formulations., (© Georg Thieme Verlag KG Stuttgart · New York.)

Microwave assisted extraction (MAE) followed by microplate solid phase extraction (MPSPE) coupled with ultra high performance liquid chromatography (UHPLC) for the semi-quantitative determination of colchicine, 3-demethyl colchicine and 2-demethyl colchicine in postmortem rat bone is described. Rats (n=4) received 50mg/kg colchicine (i.p), and euthanized by CO2 asphyxiation. Remains decomposed to skeleton outdoors and vertebral bones were collected cleaned, and ground to a fine powder. Powdered bone underwent MAE using methanol in a closed microwave system, followed by MPSPE and analysis using UHPLC-PDA. MAE analyte stability was assessed and found to be stable for at least 60 min irradiation time. The majority (>95%) of each analyte was recovered after 15 min. The MPSPE-UHPLC method was linear between 10 and 2,000 ng/mL, with coefficients of variation <20% in triplicate analysis, with a limit of detection of 10 ng/mL for each of the three analytes. Following MAE for 30 min (80°C, 1200W), MPSPE-UHPLC analysis of vertebral bone of colchicine-exposed rats detected colchicine (1.8-4.1 μg/g), 3-demethyl colchicine (0.77-1.8 μg/g) and 2-demethyl colchicine (0.43-0.80 μg/g) in all samples assayed., (Copyright © 2014 Elsevier B.V. All rights reserved.)

Background: Gloriosa superba produces an array of alkaloids including colchicine, a compound of interest in the treatment of various diseases. The tuber of Gloriosa superba is a rich source of colchicine which has shown anti-gout, anti-inflammatory, and anti-tumor activity. However, this promising compound remains expensive and Gloriosa superba is such a good source in global scale. Increase in yield of naturally occurring colchicine is an important area of investigation., Materials and Methods: The effects of inoculation by four arbuscular mycorrhizal (AM), fungi, Glomus mossae, Glomus fasciculatum, Gigaspora margarita and Gigaspora gilmorei either alone or supplemented with P-fertilizer, on colchicine concentration in Gloriosa superba were studied. The concentration of colchicine was determined by high-performance thin layer chromatography., Results: The four fungi significantly increased concentration of colchicine in the herb. Although there was significant increase in concentration of colchicine in non-mycorrhizal P-fertilized plants as compared to control, the extent of the increase was less compared to mycorrhizal plants grown with or without P-fertilization. This suggests that the increase in colchicine concentration may not be entirely attributed to enhanced P-nutrition and improved growth. Among the four AM fungi Glomus mossae was found to be best. The total colchicine content of plant (mg / plant) was significantly high in plants inoculated with Glomus mossae and 25 mg kg(-1)phosphorus fertilizer (348.9 mg /plant) while the control contain least colchicine (177.87 mg / plant)., Conclusion: The study suggests a potential role of AM fungi in improving the concentration of colchicine in Gloriosa superba tuber.

Because of the various matrices available for forensic investigations, the development of versatile analytical approaches allowing the simultaneous determination of drugs is challenging. The aim of this work was to assess a liquid chromatography-tandem mass spectrometry (LC-MS/MS) platform allowing the rapid quantification of colchicine in body fluids and tissues collected in the context of a fatal overdose. For this purpose, filter paper was used as a sampling support and was associated with an automated 96-well plate extraction performed by the LC autosampler itself. The developed method features a 7-min total run time including automated filter paper extraction (2 min) and chromatographic separation (5 min). The sample preparation was reduced to a minimum regardless of the matrix analyzed. This platform was fully validated for dried blood spots (DBS) in the toxic concentration range of colchicine. The DBS calibration curve was applied successfully to quantification in all other matrices (body fluids and tissues) except for bile, where an excessive matrix effect was found. The distribution of colchicine for a fatal overdose case was reported as follows: peripheral blood, 29 ng/ml; urine, 94 ng/ml; vitreous humour and cerebrospinal fluid, < 5 ng/ml; pericardial fluid, 14 ng/ml; brain, < 5 pg/mg; heart, 121 pg/mg; kidney, 245 pg/mg; and liver, 143 pg/mg. Although filter paper is usually employed for DBS, we report here the extension of this alternative sampling support to the analysis of other body fluids and tissues. The developed platform represents a rapid and versatile approach for drug determination in multiple forensic media., (Copyright © 2013 John Wiley & Sons, Ltd.)

The present work describes validated high-performance liquid chromatography (HPLC) and high-performance thin-layer chromatography (HPTLC) methods for the simultaneous determination of colchicine and khellin. The isocratic reversed-phase HPLC separation was performed on a 5 µm C18 column (Luna, Phenomenex, Torrance, CA). Good resolution between colchicine and khellin was achieved using a mixture of acetonitrile-10 mM NaH(2)PO(4) (pH 3.0, 35:65 v/v) as a mobile phase. Quantitation was achieved with ultraviolet detection at 245 nm based on peak area. The HPTLC separation was conducted on Merck HPTLC aluminum sheets of silica gel 60 F254 as stationary phase using methylene chloride-methanol (95:5 v/v) as a mobile phase. Quantification was also achieved using densitometric measurements at 245 nm. Both methods revealed reasonable validation parameters concerning selectivity, linearity, accuracy, precision and limits of detection and quantitation.

Colchicine, a natural pseudo-alkaloid found in plants such as the Colchicum autumnale and Gloriosa superba has tremendous medicinal properties, but if misused by an unqualified person can result in fatal consequences. We report a case of colchicine poisoning in an adult man as a result of consumption of the herb G. superba by a 50-year-old man and review the literature.

GC-MS analysis of Iphigenia stellata Blatter in methanol extract revealed the presence of twenty chemical compounds in corm, nine in capsule wall, seven in leaves and six in seeds. Amongst the different phytochemicals identified 2-furan-carboxaldehyde-5-(hydroxymethyl) (38% in corms), glycerine (35.4% in seeds) and n-Hexadecanoic acid (31.5% in leaves, 28% in capsule wall) were significant. Iphigenia stellata is known for the presence of colchicine. However, in the methanol extract it was not detected.

A simple, reproducible and efficient reverse phase high performance liquid chromatographic method was developed for simultaneous estimation of etoricoxib and thiocolchicoside in combined tablet dosage form. Formulation containing etoricoxib and thiocolchicoside is used as analgesic. Chromatography was performed on a 250 mm x 4.6 mm, 5-microm particle size, BDS Hypersil C-18 column with trifluoroacetic acid buffer (pH 2.6) and acetonitrile (75:25, v/v) as a mobile phase. The detection of the combined dosage form was carried out at 220 nm and a flow rate employed was 1.5 mL/min. The retention times were 6.6 and 3.1 min for etoricoxib and thiocolchicoside, respectively. Linearity was obtained in the concentration range 20 to 160 ppm for etoricoxib and in the range 2 to 16 ppm for thiocolchicoside with a correlation coefficient of 0.9918 and 0.9994, respectively. The results of the analysis were validated statistically and recovery studies confirmed the accuracy and precision of the proposed method.

An electrochemical sensor, based on a graphene oxide (GO)-Nafion composite film modified glassy carbon electrode (GCE), was developed and used for detection of trace amounts of colchicine. Owing to the large surface area, good conductivity of GO and good affinity of Nafion, the sensor exhibited excellent electrocatalytic activity for the oxidation of colchicine, displaying a wide linear response from 5.0 × 10(-8) to 2.0 × 10(-5) mol L(-1) and a low detection limit of 1.5 × 10(-8) mol L(-1) in 0.1 mol L(-1) H(2)SO(4) solution. And this modified electrode exhibited a superior immunity from epinephrine, dopamine and ascorbic acid interference. The method was also applied successfully to detect colchicine in medicinal tablets.

Colchicum autumnale is commonly known as autumn crocus, and as 'gowri gedde' in the southern region of Karnataka State in South India. It contains an alkaloid called colchicine, which blocks the cell division by inhibiting mitosis. We present a sporadic case of suicidal plant poisoning wherein a 24-year-old man consumed 'gowri gedde' to end his life. Initially he presented with severe vomiting, diarrhoea and epigastric pain. He died on the third day of ingestion due to multiorgan failure. Chemical analysis of blood and viscera obtained postmortem confirmed the presence of colchicine. Colchicine poisoning is potentially life threatening because of its high toxicity and unavailability of specific antidotal treatment. It classically presents with gastroenterocolitis, and may result in multiorgan failure in fatal cases., (Copyright © 2011 Elsevier Ltd and Faculty of Forensic and Legal Medicine. All rights reserved.)

As the most prevalent route of delivery, oral administration has the challenge of potentially low bioavailability in part because P-glycoprotein (P-gp) in the intestinal tract affects absorption. Therefore, absorption enhancers or P-gp inhibitors are strategies to solve this problem. The aim of the present study was to investigate the effects of borneol on transportation of colchicine and rhodamine123, two P-gp substrates, in rats. In vitro transportation was assessed with a diffusion chamber system with isolated rat intestines. Different concentrations of borneol (10, 40 and 80 μg/mL) were prepared in solutions with two P-gp substrates compared with blank solutions. The in vivo effects on colchicine were assessed by a pharmacokinetic study. Borneol enhanced the absorptive transport of two P-gp substrates, which was relevant to the concentration. A pharmacokinetic study showed that in the presence of borneol, a significant increase in C(max) and AUC(0→8) of colchicine occurred when compared to colchicine alone. The study showed that borneol affected two P-gp substrates in the intestine, possibly by inhibiting the effects of P-gp and enhancing intestinal absorption of drugs. Therefore, borneol could be developed as a P-gp inhibitor and absorptive enhancer.

A novel approach based on ionic liquid-single-drop microextraction (IL-SDME) online coupling with capillary electrophoresis (CE) was used to determine a toxic alkaloid--colchicine. The IL-SDME procedure was optimized by extraction solvent, drop volume controlling, sample volume and pH, extraction time, and ionic strength. Under optimum conditions, enrichment factor was as much as 41-fold with a relative standard deviation of 2.8% (n = 3). Linear range of response was observed from 1 to 100 μg/mL, with detection limit of 0.25 μg/mL and correlation coefficient (R(2) ) of 0.9994. The extraction of colchicine from spiked Lanzhou lily sample was performed and obtaining good result with an average recovery rate of 102.4 and 98.8% at 5 and 50 μg/mL, respectively. Comparing with the previous methods, IL-SDME-CE is really a convenient, economical, and environmentally benign way for determining colchicine., (Copyright © 2011 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Sensitive and accurate high-performance liquid chromatographic methods have been developed for the simultaneous determination of thiocolchicoside (TC)-glafenine (GF) (Mix I) and thiocolchicoside-floctafenine (FN) (Mix II) in their pharmaceutical formulations. The analysis for both mixtures was performed using 250 mm × 4.6 mm i.d., 5 μm particle size C18 Waters Symmetry column. The mobile phase consisted of methanol-0.035 M phosphate buffer (50:50, v/v) of pH 4.5 for Mix I and methanol-0.03 M phosphate buffer (70:30, v/v) of pH 4 for Mix II with flow rate of 1 mL/min and UV detection at 400 nm in both cases. The calibration plots were rectilinear over the concentration range of 0.2-2 μg/mL for TC in both mixtures and 20-200 μg/mL for each of GF and FN . The limits of detection for TC and GF were 0.05 μg/mL and 0.62 μg/mL, respectively, and for TC and FN were 0.02 μg/mL and 0.70 μg/mL, respectively. Additionally, the proposed methods were successfully applied to their combined tablets with average percentage recoveries of 100.35 ± 0.61 and 100.57 ± 0.72% for TC and GF respectively and for TC and FN the percentage recoveries were 101.2 ± 0.72 and 100.36 ± 0.67%, respectively. The results obtained were favorably compared with those given using the comparison methods.

Gloriosa superba is one of the poisonous plants growing in Japan. It contains potent alkaloid such as colchicine which binds to tubulin and prevents it from forming microtubules that are part of the cytoskeleton in human cells. Ingestion of Gloriosa superba tubers causes severe and potentially fatal toxic effects. We report here a case of colchicine intoxication caused by tubers of Gloriosa superba. A 58-year-old male ingested about 25 g of Gloriosa superba tubers by mistake. He believed that it was wild yam. He developed abdominal pain, vomiting and diarrhea 30 minutes after the ingestion of the plant. Forty five hours later he was taken to the emergency department. Unfortunately he died due to progressive multiple organ failure about one hour after the admission. It was two days after Gloriosa superba ingestion. The clinical features and the aspect of the poisonous plants should be acquainted with people.

A new, simple HPTLC method for determination of etoricoxib (ETO) and thiocolchicoside (THIO) in combined tablet dosage form has been developed and validated. The pharmaceutical dosage form used in this study was Nucoxia-MR tablets. Sample solutions were prepared at concentrations of 25 and 20 microg/mL for ETO and THIO, respectively. The separation was carried out on 20 x 10 cm Merck aluminum sheets precoated with a 250 microm layer of silica gel 60F254 using ethyl acetate-methanol (8 + 2, v/v) as the mobile phase. The calibration curve was linear over a range of 50-250 and 100-500 ng/band for ETO and THIO, respectively. Quantitative determination was done by densitometric scanning of bands at 290 nm. LOD and LOQ values were 10.993 and 33.314 ng/band, respectively, for ETO and 25.133 and 76.161 ng/band, respectively, for THIO. The method was validated with respect to linearity, accuracy, precision, and robustness in accordance with the International Conference on Harmonization guidelines. The method has been successfully applied to the analysis of drugs in the pharmaceutical formulation.

A rapid approach has been developed for screening trace level compounds with antitumor activities based on their interactions with microtubules. This interaction can be quantified with liquid chromatography (LC) by measuring the difference of bioactive compound's concentration before and after the formation of compound-microtubule complexes in the fast dialyzers. To test the effectiveness of this approach, several antitumor drugs such as colchicine, taxol, daunorubicin, and a non-antitumor reagent ketoprofen were used. Results indicate that the antitumor constituents can be identified without any disturbance, and the inactive components can be excluded. This screening method was then successfully applied to some potential antitumor compound mixtures, and the active compounds could be separated and screened rapidly. The binding activities measured were consistent with their cytotoxicity assays. This integrative approach is rapid and convenient for screening, isolating, and analyzing potential antitumor active compounds from a mixture.

A 65 year old woman had collected some plant leaves, cooked them in water and ate this mash with honey. A few hours after consumption first symptoms of intoxication like sickness, stomach aches and diarrhoea appeared. Her condition deteriorated, she was admitted to hospital, became comatose and finally died due to a multiorgan failure. The plants she had collected were identified as autumn crocus (Colchicum autumnale). Colchicine-concentrations detected in blood samples (HPLC/DAD) taken in hospital approximately 24 h after ingestion were 50 microg/l, in post-mortem femoral blood 65 microg/l colchicine were found (toxic range: >24 microg/l). Also all tested organs contained equally high colchicine levels. Histological examination of the liver showed diffuse vacuolization in the cytoplasma of hepatocytes. Possibly due to the relatively short survival time, only isolated mitotic structures were found within the epithelium of the colon.

Objective: In order to provide a theoretical foundation for the medically effective ingredient-based selection of elite cultivars in Comnus officinalis, a study has been conducted on the variation in medicinal effective ingredients of the fruit from the genuine producing areas, the correlation among effective medicinal ingredients and the correlation between effective ingredients and fruit shape as well as nutritional indexes., Method: The completely mature fruit was collected from the genuine producing areas Chunan county and Lin'an city of Zhejiang province. The contents of colchicine, ursolic acid and oleanolic acid were determined by HPLC, and vertical diameter/transversal diameter of the fruits, soluble solid matter and percentage of fresh flesh to the fruit were also measured., Result: (1) Ursolic acid, oleanolic acid, and colchicine in fruits ranged from 0.1010% to 0.4786%, 0.0149% to 0.1274% and 0.59% to 2.30%, respectively, and their RSD were 34.33%, 40.48% and 28.50%, respectively. (2) The correlation between effective ingredients and that between effective ingredients and fruit shape as well as nutritional indexes were as follows: the content of ursolic acid was significantly correlated with that of oleanolic acid with a correlation coefficient of 0.9796; both ursolic acid and oleanolic acid were in significantly negative correlation with soluble solid matter with a correlation coefficient of -0.5544 and -0.5118, respectively; colchicine was significantly associated with soluble solid matter with a correlation coefficient of 0.2412; colchicine, ursolic acid and oleanolic acid were in significantly negative correlation with the percentage of fresh flesh with a correlation coefficient of -0.2507, -0.2443 and -0.2406, respectively; three effective ingredients showed no correlation with the ratio of vertical diameter to transversal diameter of the fruit., Conclusion: There is a significant difference in effective ingredients among individual trees, which means that there is a big potential for selection of cultivars. Individual tree-based selection should be mainly adopted when effective ingredients are used as a main index in selection on the basis of the correlation among effective ingredients and that between effective ingredients and fruit shape as well as nutritional indexes, while ursolic acid could be combined with oleanolic acid to be used as an index and a preliminary screen could be conducted using soluble solid matter.

Implementation of an uncomplicated SPE process for the rapid extraction and preconcentration of the alkaloids, colchicine, strychnine, aconitine, and nicotine, from water, apple juice, and nonfat milk samples is presented. When coupled to analysis via micellar EKC (MEKC), the total analysis time per sample was less than 15 min for the water and juice samples and less than 20 min for the milk. The SPE process allowed for anywhere from a three to a fourteen-fold improvement in the LOD for each alkaloid when compared to detecting the alkaloids in a nontreated water sample matrix. Following SPE, the LODs for colchicine, strychnine, and nicotine were sufficient to meet levels from 150 to 5000 times more dilute than the LD(50) for a 50 kg individual drinking 12 oz of a contaminated beverage. Aconitine, on the other hand, was detected at approximately the LD(50) level. The percent recoveries for the SPE ranged from 37% to as high as 99%. Nicotine attained the highest recovery efficiencies, followed by colchicine, and finally, aconitine and strychnine, which were nearly identical. The greatest recovery efficiencies were achieved from apple juice and water, whereas nonfat milk yielded the lowest.

As a part of a project designed to investigate Colchicum species in Jordan, the chemical constituents of Colchicum crocifolium Boiss. (Colchicaceae) were investigated using LC-MS and LC-UV/Vis PDA. A decision tree for working with colchicinods has been developed by incorporating data from LC-UV/PDA and LC-MS. This dereplication strategy draws upon the UV/PDA spectra to classify compounds into one of four structural groups and combines this with retention time and mass spectra/molecular weight to identify the compounds. This strategy was applied on a small amount of extract (2 mg) of Colchicum crocifolium to dereplicate 10 known compounds from four different structural groups, namely (-)-demecolcine, 2-demethyl-(-)-colchicine or 3-demethyl-(-)-colchicine, N-deacetyl-(-)-colchicine, (-)-colchiciline, (-)-colchicine, beta-lumidemecolcine, 2-demethyl-beta-lumicolchicine or 3-demethyl-beta-lumicolchicine, N,N-dimethyl-N-deacetyl-beta-lumicornigerine, (-)-isoandrocymbine and (-)-autumnaline. Furthermore, a new compound was identi?ed as N,N-dimethyl-N-deacetyl-(-)-cornigerine. Three compounds, which had molecular ions at m/z 325, 340 and 374, could not be dereplicated into any obvious structural classes that have been isolated in our laboratories previously or reported in the literature.

Miniaturized electrochemical graphite based sensor for the analysis of colchicine, along with its detailed construction was described. The electrochemical behavior of colchicine, both by cyclic and differential pulse voltammetry was investigated, showing an irreversible reduction and oxidation mechanism. The effect of several different electrochemical mediators was studied in order to decrease the oxidation potential of colchicine, but none of them showed a favorable electron transfer between the alkaloid and the electrode's surface. The influences of different biomolecules (calf thymus DNA, bovine serum albumin and beta-tubulin) over colchicine's electrochemical behavior were also presented. For quantitative determinations the anodic differential pulse voltammetric method in H(3)PO(4)/HClO(4) 0.01 M (pH 2.05) was selected, evaluating one of the oxidation peaks of colchicine at +970 mV versus Ag pseudo-reference. The electrochemical scanning parameters were optimized by an experimental design using response surface modelling. The method was validated according to ICH guidelines in the concentration range 85-1200 ng mL(-1) (R(2)=0.9966, n=7) colchicine in terms of linearity, accuracy, limits of detection (LOD) (41 ng mL(-1)), limits of quantification (LOQ) and fidelity and it was successfully applied to the determination of colchicine in tablets, without the interference of the excipients. The studied method showed the same sensitivity as the more complex HPLC procedure and micellar electrokinetic chromatography with spectrophotometric detection.

Three novel conjugation metabolites of colchicine were identified in rat bile facilitated by enhanced on-line liquid chromatography-accurate radioisotope counting. The known 2- and 3-demethylcolchicines (DMCs) underwent O-sulfate conjugation in addition to the previously described O-glucuronidation. 2-DMC was preferably O-glucuronidated, whereas 3-DMC predominantly yielded O-sulfation conjugates, indicating phase II conjugation regiopreferences. Moreover, M1 was identified as a novel glutathione conjugate and a possible biotransformation pathway for its formation was proposed. The known 2-DMC (M6), 3-DMC (M7), 2-DMC glucuronide (M4), and novel 3-DMC sulfate (M3) were confirmed as the major metabolites. Radiometric data were acquired by the XFlow liquid chromatography-accurate radioisotope counting (XFlow LC-ARC) system, a novel technology for dynamic control of both on-column and postcolumn high-performance liquid chromatography flow rates to maximize sensitivity and resolution of radiochromatograms. A comparative evaluation was also performed between the XFlow LC-ARC system and a conventional flow radiometric detection system using bile samples from an in vivo disposition study of colchicine in male Sprague-Dawley rats. Results demonstrated a 20-fold sensitivity improvement of the XFlow LC-ARC system in comparison with radioactivity detection by conventional flow scintillation analyzers. The dynamic flow mode also provided the best chromatographic resolution. Unambiguous metabolite identification was performed by high-resolution mass spectrometry and nuclear magnetic resonance analysis.

A microchip device is demonstrated for the electrophoretic separation and UV-absorbance spectral detection of four toxic alkaloids: colchicine, aconitine, strychnine, and nicotine. A fused-silica (quartz) microchip containing a simple cross geometry is utilized to perform the separations, and a miniature, fiber-optic CCD spectrometer is coupled to the microchip for detection. Sensitive UV-absorbance detection is achieved via the application of online preconcentration techniques in combination with the quartz microchip substrate which contains an etched bubble-cell for increased pathlength. The miniature CCD spectrometer is configured to detect light between 190 and 645 nm and LabView programming written in-house enables absorbance spectra as well as separations to be monitored from 210 to 400 nm. Consequently, the configuration of this microchip device facilitates qualitative and quantitative separations via simultaneous spatial and spectral resolution of solutes. UV-absorbance limits of quantification for colchicine, 20 microM (8 mg/L); strychnine, 50 microM (17 mg/L); aconitine, 50 microM (32 mg/L); and nicotine, 100 microM (16 mg/L) are demonstrated on the microchip. With the exception of aconitine, these concentrations are > or =20-times more sensitive than lethal dose monitoring requirements. Finally, this device is demonstrated to successfully detect each toxin in water, skim milk, and apple juice samples spiked at sublethal dose concentrations after a simple, SPE procedure.

The ethyl acetate-soluble fraction from a MeOH extract of the roots of Ferula varia gave six new sesquiterpene lactones (1-6) and five known sesquiterpenes (7-11). Their structures were established on the basis of spectroscopic evidence. The cytotoxic activities of 1-11 were evaluated against selected human cancer cell lines. Compound 4 showed significant selective cytotoxicity against multidrug-resistant cancer cells (KB-C2). The cytotoxicities of compounds 1, 3, 5, 8, and 11 against KB-C2 cells were enhanced in the presence of nontoxic concentrations of colchicine.

In our continuing attempt to investigate Jordanian Colchicum species as a potential and viable source of colchicinoids; the distributions of the most interesting colchicinoid alkaloids (-)-demecolcine and (-)-colchicine in different plant parts of wildly growing C. crocifolium Boiss., C. ritchii R. Br., and C. triphyllum Kunze, were analyzed. The method is based on the use of external reference standards and a reversed-phase gradient HPLC. While (-)-colchicine was found in all plant parts of the three investigated Colchicum species, (-)-demecolcine was not detected in the leaves and daughter corms of C. triphyllum, the corms of C. crocifolium, and the flowers, stems, and mother and daughter corms of C. ritchii. C. triphyllum was found to be the highest in total (-)-colchicine content of 0.10% (wt/wt), while C. crocifolium was the highest in total (-)-demecolcine content of 0.09% (wt/wt). During flowering, leaves and corms in C. crocifolium, and leaves and stems in C. ritchii are the main storages of (-)-colchicine. (-)-Colchicine showed a homogenous distribution in all plant parts (stems, leaves, and mother and daughter corms) of C. triphyllum of 0.10% (wt/wt). Regarding (-)-demecolcine, stems and leaves of C. crocifolium, stems of C. triphyllum, and leaves of C. ritchii are the main storages. This work reports for the first time the presence of (-)-colchicine and (-)-demecolcine in C. crocifolium.

Poisoning by colchicine may occur following ingestion of this alkaloid used for the treatment of acute gouty arthritis. The authors report two fatalities and describe a liquid chromatography-electrospray ionization-tandem mass spectrometry (LC-ESI-MS-MS) triple-quadrupole method for the determination of colchicine in autopsy samples. One milliliter of heart blood, femoral blood, urine, bile, gastric, and vitreous each were extracted with saturated NH4Cl at pH 9.6 and dichloromethane/5% isopropanol. Separation was achieved on a C18-Xterra column with a mobile phase consisting of 2 mM ammonium formate buffer (pH 3)/acetonitrile in a gradient mode. Four product ions of the protonated molecule were monitored. The method was fully validated in whole blood (1 mL) and was linear in the range of 0.5-50 ng/mL (r2>0.99). The limit of detection was 0.1 ng/mL (50 times S/N), and the limit of quantitation was 0.5 ng/mL with RSDs<11.8% intraday (n=6), <18.7% interday (n=18), and accuracy<3% (n=18). Case #1: a 33-year-old nurse committed suicide by the ingestion of 80 colchicine 1-mg tablets. She died 61 h later after resuscitation procedures. Colchicine was found in heart blood at 5.2 ng/mL, femoral blood at 17.4 ng/mL, urine at 19.4 ng/mL, bile at 42.8 ng/mL, gastric at 348 ng/mL, and vitreous at 3 ng/mL. Case #2: a 57-year-old man with gout was found dead at home. Colchicine was found in heart blood at 22.8 ng/mL, femoral blood at 21.9 ng/mL, lung blood at 45.2 ng/mL, urine at 148.5 ng/mL, bile at 1818.5 ng/mL, gastric at 219.8 ng/mL, and vitreous at 0.5 ng/mL. These results were consistent with death. Because of its good sensitivity, this LC-ESI-MS-MS triple-quadrupole method is suitable for the determination of colchicine not only in fatalities but also for pharmacokinetic studies.

As part of our continuing investigation of Jordanian Colchicum species, (-)-colchicine content in C. brachyphyllum Boiss. & Haussk. ex Boiss and Colchicum tunicatum Feinbr (Colchicaceae), growing wild in Jordan, was determined during different growth stages. Using external reference standard, a reverse-phase gradient photo-diode array high performance liquid chromatography (PDA-HPLC) method was adapted. Underground parts in both species and during different growth stages, always showed higher (-)-colchicine content than the above ground parts. In C. brachyphyllum total (-)-colchicine content of underground parts during flowering stage was found to be about 0.15% (wt/wt), while that of aerial parts was only about 0.04% (wt/wt). In C. tunicatum total (-)-colchicine content of underground parts was found to be 0.12% (wt/wt), and 0.13% (wt/wt) during flowering and vegetating stages, respectively, while that of aerial parts was only about 0.04% (wt/wt) and 0.02% (wt/wt), respectively. In C. tunicatum, stems, roots and unripe seeds are the main storages of (-)-colchicine at flowering, vegetating and seeding stages, respectively, while in C. brachyphyllum, corms are the main storage of (-)-colchicine at flowering and seeding stages.

The electrochemical behavior of colchicine at an acetylene black-dihexadecyl hydrogen phosphate (denoted as AB-DHP) composite film coated glassy carbon electrode (GCE) was investigated using cyclic voltammetry (CV), linear sweep voltammetry (LSV) and differential pulse voltammetry (DPV). Compared with the poor electrochemical signal at the unmodified GCE, the electrochemical response of colchicine at the AB-DHP film modified GCE was greatly improved, as confirmed from the significant peak current enhancement. The remarkable peak current enhancement indicates that the AB-DHP modified GCE has great potential in the sensitive determination of colchicine. Thus, all the experimental conditions, which influence the electrochemical response of colchicine, were studied and the optimum conditions were achieved. Finally, a sensitive and simple voltammetric method with a good linear relationship in the range of 1.0 x 10(-7) approximately 4.0 x 10(-5) mol/L, was developed for the determination of colchicine. The detection limit of colchicine was also examined and a low value of 4.0 x 10(-8) mol/L for 4-min accumulation was obtained (S/N=3). This electrode was successfully applied to detect colchicine in human urine samples.

A new class of highly fluorescent (phi(F) 0.3-0.8) low molecular weight water-soluble cholephilic compounds has been synthesized in two steps from dipyrrinones. The dipyrrinone nitrogens are first bridged by reaction with 1,1'-carbonyldiimidazole to form an N,N'-carbonyldipyrrinone (3H,5H-dipyrrolo[1,2-c:2',1'-f]pyrimidine-3,5-dione) nucleus, and a sulfonic acid group is then introduced at C(8) by reaction with concd H(2)SO(4). The resulting sulfonated N,N'-carbonyl-bridged dipyrrinones ("sulfoglows") are isolated as their sodium salts. When the alkyl substituents of the lactam ring are lengthened from ethyl to decyl, sulfoglows become increasingly lipophilic while maintaining water solubility. Low molecular weight sulfoglows were rapidly excreted intact in both bile and urine after intravenous infusion into rats, but higher molecular weight sulfoglows were excreted more selectively in bile. Hepatobiliary excretion of sulfoglows was partially, but not completely, blocked in mutant rats deficient in the multidrug-resistance associated transport protein Mrp2 (ABCC2). These observations point to the feasibility of developing simple sulfoglows with clinical diagnostic potential that are normally excreted in bile but appear in urine when hepatic elimination is impaired by cholestatic liver disease.

Colchicine, (S)-N-(5,6,7,9-tetrahydro-1,2,3,10-tetramethoxy-9-oxobenzo-(a(-heptalen-7-yl)-acetamide, is the main alkaloid contained in Colchicum autumnale (meadow saffron). There are known colorimetric, spectrophotometric, volumetric, potentiometric, voltametric, gravimetric and various chromatographic methods for quantitative determination of colchicine, each of them presenting a series of advantages and disadvantages. As an alternative, we proposed the use of a densitometric determination for colchicine allowing the determination of this alkaloid from pharmaceutical products, as well from seeds of meadow saffron. The total alkaloid extract was separated by Thin-Layer Chromatography using Silicagel 60F(254) layers and a mixture of chloroform:acetone:diethylamine (5:4:1) as mobile phase. The same conditions were used for the determination from pharmaceutical products. Densitometric measurements were carried out at the absorption maximum (350 nm) of colchicine, the determinations being made by reflectance and by fluorescence. The peaks were optimized regarding to their area and shape by varying four scanning parameters (slit width and height, number of measurements and scanning speed). We established the calibration plot using pure colchicine in the range 50-600 ng mL(-1). The proposed method could be widely used in the pharmaceutical industry for the quick and accurate quantitative determination of colchicine because it eliminates the interferences given by other bioactive or degradation compounds. The method was characterized by validation parameters (linearity, accuracy, fidelity, sensitivity) and it was established its performances in comparison with an HPLC method and an official quantitative determination from the Romanian Pharmacopoeia X edition respectively.