Colchicine In Plasma Research Articles

In-syringe dispersive solid phase filter extraction cleanup followed by liquid chromatography-triple quadrupole mass spectrometry for fast determination of colchicine in plasma/urine

As an effective treatment for acute gouty arthritis and cardiovascular disease, colchicine is also a toxic alkaloid and may cause poisoning or even death in overdose. The study of colchicine elimination and the diagnosis of poisoning etiology need the rapid and accurate quantitative analysis method in biological matrix. An analytical method was developed for colchicine in plasma and urine by in-syringe dispersive solid phase extraction (DSPE) followed by liquid chromatography-triple quadrupole mass spectrometry (LC-MS/MS). Sample extraction and protein precipitation were proceeded with acetonitrile. The extract was cleaned by in-syringe DSPE. An XBridge™ BEH C18 column（100 mm × 2.1 mm, 2.5 µm）was used to separate colchicine by gradient elution with mobile phase of 0.01% (v/v) ammonia-methanol. The amount and filling sequence of magnesium sulfate (MgSO4) and primary secondary amine (PSA) suitable for in-syringe DSPE were studied. Scopolamine was screened as the quantitative internal standard (IS) for colchicine analysis according to the consistency of recovery rate, chromatographic retention time and matrix effects. The limits of detection for colchicine in plasma and urine were both 0.06 ng mL−1 and the limits of quantitation were both 0.2 ng mL−1. The linear range was 0.04 – 20 ng mL−1 (Equivalent to 0.2–100 ng mL−1 in plasma or urine) with a correlation coefficient r > 0.999. By IS calibration, the average recoveries at three spiking levels in plasma and urine were 95.3–102.68% and 93.9–94.8% with the relative standard deviations (RSDs) of 2.9–5.7% and 2.3–3.4%, respectively. The matrix effects, stability, dilution effects and carryover for determination of colchicine in plasma and urine were also evaluated. The elimination of colchicine within 72–384 h post-ingestion was studied for a poisoning patient with the doses of 1 mg d−1 for 39 days and then 3 mg d−1 for 15 days).

Validation of Colchicine Assay Method for Therapeutic Drug Monitoring in Human Plasma

Colchicine (COL) reduces the frequency of attacks in Familial Mediterranean Fever (FMF) patients and is effective in preventing and arresting renal amyloidosis in most patients. COL has a narrow therapeutic window. The blood concentration to achieve therapeutic effects can be determined by Therapeutic Drug Monitoring (TDM). However, the use of selective and sensitive analytical methods is necessary for achieving success with TDM. The purpose of this study is to develop and validate a new method for quantitative assay of COL in human plasma samples by liquid chromatograph- tandem mass spectrometry (LC-MS/MS). In our study, to 1ml plasma sample, 0.25 ml internal standard solution was added. The solution was extracted by liquid-liquid extraction (LLE). The method was validated according to the European Medicines Agency (EMEA). The total run time was 8 min in LC-MS/MS. The method has been validated over the 0.25 - 8.0 ng/mL calibration range for COL. It was seen that the method has been validated since the results of the analysis meet the EMEA criteria. In our study, COL plasma levels were found to be approximately 1.097±0.42 ng/ml in 40 FMF patients using an oral dose of 1.5-2 mg/day. A validated, rapid, simple, cost-effective, and sensitive LC-MS/MS method was developed and optimized for quantitation of COL in plasma. It has been thought that pharmacokinetic studies of COL in plasma can be performed easily using this validated method

Simultaneous determination of colchicine and febuxostat in rat plasma: Application in a rat pharmacokinetic study.

A selective, sensitive and rapid LC-MS/MS method has been developed and validated as per US Food and Drug Administration regulatory guidelines for the simultaneous quantitation of colchicine and febuxostat in rat plasma. Colchicine and febuxostat were extracted from the rat plasma using 10% tert-butyl methyl ether in ethyl acetate using colchicine-d6 as an internal standard (IS). The chromatographic separation of colchicine, febuxostat and the IS was achieved using a mobile phase comprising 5 mm ammonium formate and 0.025% formic acid in acetonitrile (20:80, v/v) in isocratic mode on an Eclipse XDB-C18 column. The injection volume and flow rate were 5.0 μl and 0.9 ml/min, respectively. Colchicine and febuxostat were detected by positive electrospray ionization in multiple reaction monitoring mode using transition pairs (Q1 → Q3) of m/z 400.10 → 358.10 and 317.05 → 261.00, respectively. The assay was linear in the ranges of 0.25-254 and 2.60-622 ng/ml for colchicine and febuxostat, respectively. The inter- and intra-day precision values were 0.58-13.0 and 1.03-4.88% for colchicine and febuxostat, respectively. No matrix or carryover effects were observed during the validation. Both analytes were stable on the bench-top, in the autosampler and in storage (freeze-thaw cycles and long-term storage at -80°C). A pharmacokinetic study in rats was performed to show the applicability of the validated method.

Sensitive method for the Determination of Colchicine in human plasma by gradient UPLC-ESI-MS/MS: separation of endogenous Interference

Sensitive method for the Determination of Colchicine in human plasma by gradient UPLC-ESI-MS/MS: separation of endogenous Interference

LC-MS/MS quantification of free and Fab-bound colchicine in plasma, urine and organs following colchicine administration and colchicine-specific Fab fragments treatment in Göttingen minipigs.

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.

Highly Sensitive Determination of Colchicine in Human Plasma by UPLC-MS/MS for a Clinical Study in Healthy Subjects

Highly Sensitive Determination of Colchicine in Human Plasma by UPLC-MS/MS for a Clinical Study in Healthy Subjects

Rugged and Accurate Quantitation of Colchicine in Human Plasma to Support Colchicine Poisoning Monitoring by Using Turbulent-Flow LC–MS/MS Analysis

Colchicine is a common drug used in inflammatory diseases. The narrow therapeutic index requires fast and reliable techniques for its quantitation. An online, automated sample preparation using TurboFlow™ technology combined with triple-stage quadrupole MS detection was applied to identify colchicine in human plasma and follow intoxications. Plasma samples (200 µl) were mixed with deuterated colchicine and protein precipitation ZnSO4 solutions. After centrifugation, supernatants were extracted onto a Cyclone P TurboFlow column and eluted onto a narrowbore Hypersil™ GOLD column with a methanol/water gradient. Analytes were monitored in SRM mode (positive electrospray). Total run time was 9.5 min. Calibration curves ranged from 0.342 to 17.1 ng/ml, with significant linearity (R(2) >0.99). Inter- and intra-assay precisions were <16.8% and accuracy was 84.4-110%. This method is suitable for monitoring intoxication in patients undergoing chronic treatment and is routinely applied to toxicological samples.

Determination of Colchicine in Mouse Plasma by High Performance Liquid-chromatographic Method with UV Detection and Its Application to Pharmacokinetic Studies

A simple and sensitive high performance liquid chromatography method with UV detection was described for the determination of colchicine (COL) in mouse plasma. After single-step deproteinization by acetonitrile using berberine hydrochloride as an internal standard (I.S.), solutes were separated on a Diamonsil C(18) column (250 mm x 4.6 mm I.D., 5 microm particle size) (Dikma), using acetonitrile-0.15% phosphoric acid solution (27:73, v/v) as mobile phase (flow-rate 1.0 ml/min); wavelength of the UV detector was set at 350 nm. No interference from any endogenous substances was observed during the elution of COL and internal standard (I.S., berberine hydrochloride). The retention times for COL and I.S. were 11.23 min and 8.82 min, respectively. The limit of quantification was evaluated to be 1.5 ng/ ml and the limit of detection was 0.5 ng/ml. The method was used in the study of pharmacokinetics of COL after intravenous injection (i.v.) and intraperitoneal injection (i.p.). The result indicated that COL disappears from the plasma according to a three compartment open model.

Rapid and sensitive liquid chromatography–tandem mass spectrometry method for the quantitation of colchicine in human plasma

A rapid and sensitive method to determine colchicine in human plasma by liquid chromatography–tandem mass spectrometry (LC–MS/MS) has been developed. Colchicine and the internal standard (I.S.), tegafur, were extracted from the matrix with n-hexane:dichloromethane:isopropanol (300:150:15, v/v/v) and separated by reversed-phase high-performance liquid chromatography (HPLC) using formic acid:10 mM ammonium acetate:methanol (1:49:75, v/v/v) as the mobile phase in a run time of 2.5 min. Detection was carried out by electrospray positive ionization mass spectrometry in the multiple-reaction monitoring (MRM) mode. The assay was linear in the concentration range 0.050–10 ng/ml with intra- and inter-day precision (as relative standard deviation (R.S.D.)) of <2 and <7%, respectively. The method was applied to a pharmacokinetic study of colchicine in healthy volunteers given an oral dose of 2.0 mg.

A Novel LC-ESI-MS-MS Method for Sensitive Quantification of Colchicine in Human Plasma: Application to Two Case Reports

A novel method based upon liquid chromatography coupled to ion trap mass spectrometry (MS) detection with electrospray ionization interface has been developed for the identification and quantification of colchicine in plasma or whole blood. Colchicine was isolated from plasma using a liquid-liquid extraction with dichloromethane at pH 8.0 and embutramide as an internal standard, with satisfactory extraction recoveries. Solutes were separated on a 3-microm C18 Uptisphere (Interchim) column (150 x 2.0-mm i.d.) using acetonitrile/2 mM NH4COOH pH 3.8 buffer (50:50, v/v) as the mobile phase with a flow-rate of 200 microL/min. Data were collected either in full-scan MS mode at m/z 100-450 or in full-scan MS-MS mode, selecting the ion m/z 400.1 for colchicine and m/z 294.1 for embutramide. The most intense daughter ion of colchicine (m/z 358.1) and embutramide (m/z 207.9) were used for quantification. Retention times were 2.40 and 4.25 min for colchicine and embutramide, respectively. Calibration curves were linear in the 0.50-50 ng/mL range. The limits of detection and quantification were 0.05 ng/mL and 0.50 ng/mL, respectively. The intra- and interassay precisions were < 14%, and the intra- and interassay accuracies were in the 97-105.8% range at either 2 or 20 ng/mL. A fatal case of colchicine self-poisoning with a lethal blood concentration of 60 ng/mL and nonfatal case with a plasma sample collected very late (at least 36 h after the ingestion) are presented. The described method enables the unambiguous identification and quantification of colchicine with a very good sensitivity, using only 1 mL of sample.

Optimisation of the Treatment of Acute Gout

Most of the drugs prescribed to treat acute gouty attacks were used before the introduction of modern clinical trials. Thus, there are few well-designed studies available to evaluate these drugs. Nevertheless, worldwide clinical experience supports the use of most nonsteroidal anti-inflammatory drugs (NSAIDs), colchicine and corticosteroids in the treatment of acute gout. Colchicine has been widely used but toxicity, especially gastrointestinal adverse effects, are a major concern. Therapeutic regimens involving hourly or 2-hourly administration were based on the short initial half-life of colchicine in plasma. Other therapy schedules, such as early 8-hourly administration, may be equally effective and have fewer adverse effects. Unfortunately, comparative studies to investigate this have not been performed. Colchicine should not be prescribed to patients with either severe renal insufficiency or combined hepatic-renal insufficiency. Doses should be halved in patients with moderate renal function impairment. NSAIDs are the most widely prescribed drugs in the treatment of acute gout. Few comparative data are available, but any of the most potent NSAIDs are probably useful in the control of pain and inflammatory signs of acute gouty arthritis. Pharmacokinetic properties should be taken into account when selecting an NSAID for the treatment of gout, as rapid absorption and a short half-life may help to avoid accumulation in patients with subclinical renal function impairment. Comorbidities should always be kept in mind when prescribing NSAIDs. Patients with previous or recent gastrointestinal bleeding, those receiving anticoagulant therapy or with haemorrhage diathesis, and those with renal insufficiency are at risk of developing severe adverse effects from NSAID administration. Corticosteroids are probably a reasonable choice for patients in whom colchicine and NSAIDs may be hazardous or for those with a history of previous intolerance to these drugs. Few trials using prednisone, prednisolone or triamcinolone acetonide are available, and dosages are prescribed following empirical data. Corticotropin has also been used to treat acute gout. Although it has been proven to be as effective as other corticosteroids or indomethacin, the need for multiple doses, parenteral administration and the high cost are major limitations for its use. Currently, the choice of a drug for the treatment of acute gout will depend on the balance between its efficacy and the potential adverse effects in a particular patient.

Role of P-170 glycoprotein in colchicine brain uptake

To study the role of P-glycoprotein (P-gp) in the delivery of colchicine from blood to brain, the pharmacokinetics of colchicine in plasma and brain was studied in the rat by an in vivo method and by the in situ brain perfusion technique. Colchicine was administered intravenously at three doses (1, 2.5, and 5 mg/kg) with or without an inhibitor of P-gp, verapamil (0.5 mg/kg i.v.); blood and brain samples were taken at t = 1, 2, and 3 hr. Areas under the colchicine curve at doses from 2.5 to 5 mg/kg were proportional to dose for plasma but not for brain. At a colchicine dose of 5 mg/kg, verapamil co-treated rats showed a 1.65-fold enhancement of the colchicine concentration in plasma but a 4.5-fold enhancement in brain. During short experimental times (in situ brain perfusion technique), a comparable enhancement was found (4.26-fold): mean distribution volumes of colchicine were enhanced from 0.23 +/- 0.17 to 0.98 +/- 0.19 microl/g for the eight gray areas, and no effect was observed in the choroid plexus, which do not express P-gp. These results clearly show that P-gp, present at the luminal surface of the capillary endothelial cells, is responsible for the weak penetration of colchicine into the brain.

Role of P‐170 glycoprotein in colchicine brain uptake

To study the role of P-glycoprotein (P-gp) in the delivery of colchicine from blood to brain, the pharmacokinetics of colchicine in plasma and brain was studied in the rat by an in vivo method and by the in situ brain perfusion technique. Colchicine was administered intravenously at three doses (1, 2.5, and 5 mg/kg) with or without an inhibitor of P-gp, verapamil (0.5 mg/kg IV); blood and brain samples were taken at t = 1, 2, and 3 hr. Areas under the colchicine curve at doses from 2.5 to 5 mg/kg were proportional to dose for plasma but not for brain. At a colchicine dose of 5 mg/kg, verapamil co-treated rats showed a 1.65-fold enhancement of the colchicine concentration in plasma but a 4.5-fold enhancement in brain. During short experimental times (in situ brain perfusion technique), a comparable enhancement was found (4.26-fold): mean distribution volumes of colchicine were enhanced from 0.23 ± 0.17 to 0.98 ± 0.19 μl/g for the eight gray areas, and no effect was observed in the choroid plexus, which do not express P-gp. These results clearly show that P-gp, present at the luminal surface of the capillary endothelial cells, is responsible for the weak penetration of colchicine into the brain. J. Neurosci. Res. 49:80–88, 1997. © 1997 Wiley-Liss Inc.

High-performance liquid chromatography coupled to ion spray mass spectrometry for the determination of colchicine at ppb levels in human biofluids

An original method based upon high-performance liquid chromatography coupled to ion spray mass spectrometry (HPLC-ISP-MS) has been developed for the identification and quantification of colchicine (COL) in human blood, plasma or urine. After single-step liquid-liquid extraction by dichloromethane at pH 8.0 using tofisopam (TOF) as an internal standard, solutes are separated on a 5-μm C 18 Microbore (Alltech) column (250×1.0 mm, I.D.), using acetonitrile-2 mM NH 4COOH, pH 3 buffer (75:25, v/v) as the mobile phase (flow-rate 50 μl/min). Detection is done by a Perkin-Elmer Sciex API-100 mass analyzer equipped with a ISP interface (nebulizing and curtain gas: N 2, quality U; main settings: ISP, +4.0 kV; OR, +50 V; Q0, −10 V; Q1, −13 V; electron multiplier, +2.2 kV); MS data are collected as either total ion current (TIC, m/z 100–500 or 380–405), or selected ion monitoring (SIM) at m/z 400 and 383 for COL and TOF, respectively. COL mass spectrum shows a prominent molecular ion [M+H] + at m/z 400. Increasing OR potential fails to provide a significant fragmentation. Retention times are 2.70 and 4.53 min for COL and TOF, respectively. The quantification method shows a good linearity ( r = 0.998) over a concentration range from 5 to 200 ng/ml. The lower limit of detection in SIM mode is 0.6 ng/ml COL, making the method convenient for both clinical and forensic purposes.

Radioimmunoassay for colchicine in plasma and urine.

A radioimmunoassay for the measurement of colchicine (in quantities as small as 0.05 nanogram) in plasma and urine was developed with the use of an antibody from immunized rabbits. After the intravenous injection of 2 milligrams of colchicine in seven subjects, the calculated zero-time concentration in the plasma was 2.9 +/- 1.5 micrograms per deciliter, and the mean half-time in the plasma was 58 +/- 20 minutes. Declining, but measurable, amounts of colchicine could be detected in urine up to day 9 after the drug was administered.

Purification of colchicine, its photoisomer(s), and some congeners by paper and thin-layer chromatography

Four paper and four thin-layer chromatographic systems were studied for their ability to separate colchicine from its photoisomer(s) and other congeners. The best thin-layer systems were found to be chloroform: acetone:diethylamine (5:4:1) and chloroform:diethylamine (9:1) on silica gel. The most useful paper chromatographic systems were found to be benzene:acetic acid:water (10:4:10) and benzene:hexane:acetic acid:water (6:4:3:7) run for 4 hours. The importance of identifying and quantitating the light-induced degradation products of colchicine in radioisotopic studies on the metabolic fate of this drug in the human subject was stressed in these studies. The methods described were used to provide rapid and reproducible techniques for the measurement of colchicine in plasma, urine, and white blood cells.

Colchicine plasma levels: Implications as to pharmacology and mechanism of action

A new method for quantitating colchicine in plasma is described, and plasma levels after administration in conventional therapeutic doses are reported in four groups of patients. In the first group the patients had normal renal and hepatic function and were not hyperuricemic. The second group was composed of patients with gout, the third of patients with severe renal disease and the fourth of patients with severe hepatic disease. The mean fifteen minute colchicine concentration in all groups was 1.14 ± 0.85 μg per 100 ml of plasma; the mean concentration at extrapolated zero time was 1.92 μg per 100 ml of plasma. In the patients with liver disease plasma colchicine concentrations were high and disappearance of the drug was rapid, whereas in the patients with gout and those with severe renal disease plasma concentrations were similar to those in the first group, but disappearance rates were slower. In vitro studies of others have shown that colchicine affects many leukocyte functions. Migration of polymorphonuclear leukocytes toward a chemotactic source was the only leukocytic activity affected by colchicine concentrations in the range of those found in the plasma of these patients.