Quantitative analysis of clonidine and ephedrine by a microfluidic system: On-chip electromembrane extraction followed by high performance liquid chromatography.

In this work, a microfluidic device was developed for on-chip electromembrane extraction of trace amounts of ephedrine (EPH) and clonidine (CLO) in human urine and plasma samples followed by HPLC-UV analysis. Two polymethylmethacrylate plates were used as substrates and a microchannel was carved in each plate. The microchannel channel on the underneath plate provided the flow pass of the sample solution and the one on the upper plate dedicated to a compartment for the stagnant acceptor phase. A piece of polypropylene sheet was impregnated by an organic solvent and mounted between the two parts of the chip device. An electrical field, across the porous sheet, was created by two embedded platinum electrodes placed in the bottom of the channels which were connected to a power supply. The analytes were converted to their ionized form, passed through the supported liquid membrane, and then extracted into the acceptor phase by the applied voltage. All the effective parameters including the type of the SLM, the SLM composition, pH of donor and acceptor phases, and the quantity of the applied voltage were evaluated and optimized. Several organic solvents were evaluated as the SLM to assess the effect of SLM composition. Other parameters were optimized by a central composite design. Under the optimal conditions of voltage of 74V, flow rate of 28µLmin-1, 100 and 20mM HCl as acceptor and donor phase composition, respectively, the calibration curves were plotted for both analytes. The limits of detection were less than 7.0 and 11µgL-1 in urine and plasma, respectively. The linear dynamic ranges were within the range of 10-450 and 25-500µgL-1 (r2˃0.9969) for CLO, and within the range of 20-450 and 30-500µgL-1 (r2˃0.9907) for EPH in urine and plasma, respectively. To examine the capability of the method, real biological samples were analyzed. The results represented a high accuracy in the quantitative analysis of the analytes with relative recoveries within the range of 94.6-105.2% and acceptable repeatability with relative standard deviations lower than 5.1%.

Magnetic molecularly imprinted polymer nanoparticles for selective solid phase extraction and pre-concentration of Tizanidine in human urine.

In this work, the magnetic molecularly imprinted polymer nanoparticles (MMIP-NPs) for the selective pre-concentration of Tizanidine have been described. The polymer nanoparticles were synthesized by the polymerization of methacrylic acid as a functional monomer, ethylene glycol dimethacrylate as a cross-linker, 2,2-azobisisobutyronitrile as an initiator and Tizanidine as a template molecule. The MMIP-NPs were characterized by scanning electron microscopy (SEM) and thermogravimeteric analysis (TGA). Imprinted Tizanidine molecules were removed from the polymeric structure using acetic acid in methanol (10:90 V/V%), as the eluent solvent. The limits of detection (L.O.D) for Tizanidine were 1.13×10(-6)M and 1.68×10(-6)M in ultrapure water and urine, respectively. Also, the relative standard deviations (R.S.D) in ultrapure water and urine were 2.21% and 2.58%, respectively. The method was applied to the determination of Tizanidine in the human urine samples.

Sensitive spectrofluorimetric determination of tizanidine in pharmaceutical preparations, human plasma and urine through derivatization with dansyl chloride.

A sensitive spectrofluorimetric method was developed for the determination of tizanidine in human plasma, urine and pharmaceutical preparations. The method is based on reaction of tizanidine with 1-dimethylaminonaphthalene-5-sulphonyl chloride (dansyl chloride) in an alkaline medium to form a highly fluorescent derivative that was measured at 511 nm after excitation at 383 nm. The different experimental parameters affecting the fluorescence intensity of tizanidine was carefully studied and optimized. The fluorescence-concentration plots were rectilinear over the ranges 50-500 and 20-300 ng/mL for plasma and urine, respectively, detection limits of 1.81 and 0.54 ng/mL and quantification limits of 5.43 and 1.62 ng/mL for plasma and urine, respectively. The method presents good performance in terms of linearity, detection and quantification limits, precision, accuracy and specificity. The proposed method was successfully applied for the determination of tizanidine in pharmaceutical preparations. The results obtained were compared with a reference method, using t- and F-tests.

[Analysis of clonidine in biological fluids using gas chromatography with an electron capture detector].

The method of clonidine identification in blood and urine is described. It is based on liquid-liquid extraction and purification with toluol, subsequent derivation with pentafluobenzoylchloride and test on gas chromatograph with a detector of electron capture. The method is proposed for expert examinations.

Munchausen syndrome by proxy (MSBP): an extreme form of child abuse with a special forensic challenge.

Munchausen syndrome by proxy (MSBP) is a special form of child abuse in which an adult repeatedly produces symptoms of illness in a person under his/her care. In most cases the perpetrators are mothers who repeatedly and in different ways produce or feign symptoms of illness in their children in order to obtain medical treatment for them. MSBP is thus a special form of child abuse that is also of importance in the field of forensic medicine and a particular challenge to the medicolegal expert. We report two cases of poisoning with different substances (clozapine and clonidine) detected by toxicological investigations at our Department of Legal Medicine. The relevance of the problem for the medicolegal expert and the importance of an interdisciplinary co-operation are pointed out.

Isotachophoretic determination of bisoprolol, clonidine, disopyramide and tolazoline in human fluids.

Separation and determination of bisoprolol, clonidine, disopyramide and tolazoline in control serum and in human urine was investigated by capillary isotachophoresis. The drugs were separated by using the cationic electrolyte system. viz., sodium acetate buffer (pH 4.64) (c1 = 10 mM)-beta-alanine. The compounds were almost totally isolated from serum by solid-phase extraction using a Sep-Pak C18 cartridge. The recovery of compounds varied from 87 to 99%. The linear calibration range was studied to apply the method to real human fluids. The limit of determination of the drugs was 40.0 micrograms/ml serum. The limit of determination by direct sampling for bisoprolol is 3 micrograms/ml urine.

Pharmacokinetics of transdermally delivered clonidine.

We detail a series of pharmacokinetic investigations to determine the dose linearity, the effect of site of application, the duration of steady-state plasma concentrations, and the effect of chronic application when clonidine is administered transdermally. Dose linearity was assessed in six subjects with normotension after application of increasing sizes of transdermal clonidine systems (3.5, 7.0, and 10.5 cm2 size) to the upper outer arm. Of the six subjects studied, five had linear relationships between clonidine plasma concentrations at steady state and system size of greater than 0.975; in the sixth subject the correlation was greater than 0.90. The mean steady-state plasma concentrations with 3.5, 7.0, and 10.5 cm2 systems were 0.39, 0.84, and 1.12 ng/ml, respectively. The influence of site and duration of application on the absorption of transdermal clonidine was studied in eight subjects with normotension by use of the 3.5 cm2 system. The mean steady-state plasma concentration over the time interval from 3 to 7 days after application to the arm or to the chest did not significantly differ. When a system was left on the chest or arm for a total of 11 days (4 days beyond the recommended time to change systems), the plasma concentrations of seven of eight subjects with application to the arm and of six of eight subjects with application to the chest remained constant through day 11. The influence of consecutive applications of 3.5 cm2 transdermal clonidine systems on steady-state plasma clonidine concentrations was also studied in eight subjects with normotension over an 11-day period.(ABSTRACT TRUNCATED AT 250 WORDS)

Pharmacokinetics of oral sustained release clonidine in humans.

Tablet cores of clonidine (Catapres) showing varying rates of in vitro dissolution were administered in a crossover study with intravenous clonidine to determine the rate of absorption into the systemic circulation in humans. Excellent bioavailability (0.87-0.96) was obtained in all 12 subjects for each of the formulations. When peak concentration and time to peak are compared for the standard immediate release cores and the cores designed to be slowly dissolving there was a significant difference (p less than 0.05). Absorption rate plots for each formulation in each subject were obtained using the Loo-Riegelman procedure of comparing the oral pharmacokinetic profile with the intravenous profile. As the release from the cores were formulated to be slower, the in vivo absorption rate of clonidine approached zero order input. Incorporating combinations of sustained release and immediate release cores into a capsule (Perlonget) gave predictable absorption rates, permitting the formulation scientist to control the plasma levels obtained from oral dosing of clonidine. The intravenous pharmacokinetics of clonidine after a 0.2 mg infusion followed a tri-exponential decline with a final phase half-life of 13.6 h (harmonic mean). Following the intravenous infusion 42.3% of the dose was excreted unchanged in the urine with a renal clearance of 10.05 +/- 0.65 1/h (167 ml/min).

Radioimmunoassay for clonidine in human plasma and urine using a solid phase second antibody separation.

A reliable, sensitive, and specific radioimmunoassay (RIA) procedure for the quantitation of clonidine in plasma and other biological fluids was developed. The detection limit of the assay is 2 pg based on a 200 microliters sample. Nine commonly used drugs were found not to interfere with the RIA. The utility of the assay was demonstrated in a bioavailability study of clonidine conducted with 24 healthy subjects. Clonidine was readily quantitated in plasma over 4 half-lives. This assay is suitable for pharmacokinetic and bioavailability studies as well as therapeutic drug monitoring of patients.

Biotransformation of lofexidine in humans.

1. Concentrations of unchanged drug and patterns of radioactive components in urine have been determined by h.p.l.c. following single oral doses of [14C]lofexidine hydrochloride (0.32 mg) to six human subjects. 2. A mean of 12% of the administered dose was excreted in urine as unchanged lofexidine, but the wide range (5-20% dose) indicated significant intersubject variation in the extent of biotransformation. This drug would appear to be metabolized more extensively than the related anti-hypertensive agent, clonidine. 3. The principal metabolite of lofexidine was 2,6-dichlorophenol, which was apparently excreted in urine as two O-glucuronic acid conjugates. The same two metabolites were also the main 14C components circulating in plasma at peak 14C concn. Formation of the phenol from lofexidine probably involved direct O-dealkylation rather than stepwise degradation of the side-chain. 4. Patterns of 3H components in the urine of rats and dogs after oral administration of [3H]lofexidine hydrochloride (0.1 mg/kg) were generally similar to those in human urine.

Tissue pharmacokinetics of clonidine in rats.

The antihypertensive drug clonidine exhibits nonlinear pharmacokinetics in man and rats after intravenous injection. In order to define the basis of this nonlinearity, tissue kinetics of clonidine in rats were determined at three dose levels. It was found that tissue concentrations of clonidine were linearly related to dose increases in most organs with the exception of the heart, suggesting that there was a limited binding capacity in this organ. The rate of disappearance of clonidine from most tissues was best described by a monoexponential curve with half-lives of 30 to 120 min. An exception was the stomach, and clonidine accumulated in this organ, probably due to a pH partitioning effect of this weak base. Renal clearance of clonidine in rats was also examined and found to decrease by approximately 40% when the dose was increased from 50 microgram/kg to 250 microgram/kg. It was concluded that renal clearance and and possibly fecal clearance could explain the nonlinear pharmacokinetics of clonidine.

The measurement of clonidine in human plasma and urine by combined gas chromatography mass spectrometry with ammonia chemical ionization.

The dimethyl derivatives of clonidine and [2H4]clonidine give good chemical ionization mass spectra when ammonia is used as reagent gas. A gas chromatographic mass spectrometric selected ion monitoring assay for the estimation of clonidine in plasma and urine using ammonia chemical ionization is described.

Proof of the linearity of the pharmacokinetics of alinidine in man.

The pharmacokinetics of alinidine was investigated in two groups of volunteers: Group I (N=5) received on two occasions single doses of 14C-labelled drug given orally (40 mg) or intravenously (10 mg); Group II (N=6) received single oral doses 10, 30, or 90 mg dissolved in 20 ml water. The samples from Group I were analysed by two different and independent methods (RIA and counting total radioactivity). The results obtained by the two methods were identical, since the compound was not metabolized. The plasma concentrations and renal excretion data obtained from both groups were individually fitted to an open three compartment model. Independent of the route of administration and of the doses given, similar pharmacokinetic parameters were calculated for each group and each trial. The half lives of the distribution and elimination phases were t1/2 alpha: 36-41s, t1/2 beta: 9.9-11.1 min and t 1/2 gamma: 2.7-3.8h. There was a linear relationship between the dose administered and the resulting areas under the plasma concentration curves (AUC). Following a lag period (tau =0.19-0.22h), the peak plasma concentration was reached 0.6-1.2h after oral administration. Oral alinidine was 100% bioavailable.

Determination of submicrogram quantities of clonidine in biological fluids.

A sensitive and specific GLC method using electron-capture detection was developed for clonidine in plasma and urine. Di-perfluoroacyl derivatives of both clonidine and the 4-methyl analog of clonidine (used as an internal standard) were formed, and an extraction process was developed for the removal of excess derivatization reagent and endogenous biological compounds; the assay permitted quantification of 25 pg of clonidine/ml in a 4-ml plasma sample. The assay was used to elucidate the time course of plasma concentrations in a normotensive subject following oral administration of 50, 100, and 200 microgram of clonidine hydrochloride and also to determine unchanged drug excreted in the urine.