Applications of shun shell column and nanocomposite sorbent for analysis of eleven anti-hypertensive in human plasma.

High-performance liquid chromatography (HPLC) and solid phase micro membrane tip extraction (SPMMTE) methods are developed for the simultaneous analysis of eleven cardiovascular drugs in human plasma. Iron nanoparticles were obtained by the green method, characterized by XRD, FT-IR, TEM, and EDS and utilized in SPMMTE for sample preparation. The mobile phase used was ammonium acetate buffer-methanol-acetonitrile (65:18:17) with a 1.0 mL/min flow rate at 260 nm detection. Column used was Sunshell C18 150 × 4.6 mm, 2.6 µm. The values of k, α, and Rs were ranged from 040 to109.22, 1.20 to 2.67 and 1.0 to 26.18. SPMMTE and HPLC methods were fast, reproducible, precise, robust, economic and rugged for analysis of methyldopa, hydrochlorothiazide, prazosin hydrochloride, furosemide, labetalol, propranolol, valsartan, losartan potassium, diltiazem, irbesartan and spironolactone in human plasma. The recoveries (%) of methyldopa, hydrochlorothiazide, prazosin hydrochloride, furosemide, labetalol, propranolol, valsartan, losartan potassium, diltiazem, irbesartan, and spironolactone were 91.0, 85.2, 92.3, 90.4, 90.1, 85.6, 86.6, 86.2, 85.1, 86.6, and 85.7, respectively. These results showed that SPMMTE and HPLC methods can be applied to test the described drugs in several matrices.

An Eco-Friendly Direct Injection HPLC Method for Methyldopa Determination in Serum by Mixed-Mode Chromatography Using a Single Protein-Coated Column.

A simple, rapid and environment-friendly direct injection HPLC method for the determination of methyldopa (MTD) in human serum has been developed and validated. The method was based on cleanup and separation of MTD from serum by mixed-mode liquid chromatography using a single protein-coated TSK gel ODS-80 TM analytical column (50 × 4.0 mm i.d., 5 µm). The protein-coated column exhibited excellent resolution, selectivity and functioned in two chromatographic modes: size-exclusion chromatography [i.e., solid-phase extraction (SPE) for serum proteins] and reversed-phase chromatography for the final separation of MTD. SPE and HPLC separation were carried out simultaneously with a green mobile phase consisting of acetate buffer (0.1 M, pH 2.4) at a flow rate of 1 mL/min and at room temperature (23 ± 1°C). The eluent was monitored at emission and excitation wavelengths of 320 and 270 nm, respectively. A calibration curve was linear over the range of 0.1-30 µg/mL with a detection limit of 0.027 µg/mL. This online SPE method was successfully applied to real samples obtained from patients receiving MTD therapy.

Levodopa-related cysteinyl-glycine and cysteine reduction with and without catechol-O-methyltransferase inhibition in Parkinson's disease patients.

Oxidative stress is influenced by the thiol homeostasis, which regulates the redox milieu via glutathione. Components of glutathione metabolism are cysteine and cysteinyl-glycine. Both substrates decay following levodopa application or dopamine-related oxidative stress. Objective was to investigate the impact of an acute levodopa application with and without catechol-O-methyltransferase inhibitor on cysteine- and cysteinyl-glycine plasma levels. On two investigation days, 13 patients with Parkinson's disease took one retarded release 200-mg levodopa/50 mg carbidopa-containing tablet or one 150-mg levodopa/50-mg carbidopa/200-mg entacapone formulation under standardized conditions. Levodopa, 3-O-methyldopa, cysteine and cysteinyl-glycine were measured at baseline, 80 and 140 min following levodopa administration. Cysteine and cysteinyl-glycine similarly decreased, levodopa was nearly equal during both conditions. Entacapone lowered 3-O-methyldopa. Cysteine decay may be due to an elevated glutathione generation, which consumes cysteine. Cysteinyl-glycine decrease results from the alternative glutathione transformation to its oxidized form glutathione dissulfide after free radical scavenging.

Development and validation of a high-performance liquid chromatography-electrospray ionization-MS/MS method for the simultaneous quantitation of levodopa and carbidopa in human plasma.

A sensitive and fast high-performance liquid chromatography-electrospray ionization-MS/MS method for the simultaneous quantitation of levodopa and carbidopa in human plasma was developed and validated. A simple protein precipitation step with perchloric acid was used for the cleanup of plasma, and methyldopa was added as an internal standard. The analyses were carried out using an ACE C(18) column (50 × 4.6 mm i.d.; 5 µm particle size) and a mobile phase consisting of 0.2% formic acid and acetonitrile (90:10). The triple-quadrupole mass spectrometer equipped with an electrospray source in positive mode was set up in the selective reaction monitoring mode to detect the ion transitions m/z 198.1 → m/z 107.0, m/z 227.2 → m/z 181.0, and m/z 212.1 → m/z 139.2 for levodopa, carbidopa, and methyldopa, respectively. The method was validated and proved to be linear, accurate, and precise over the range 50-5000 ng/mL for levodopa and 3-600 ng/mL for carbidopa. The proposed method was successfully applied in a pharmacokinetic study with a levodopa/carbidopa tablet formulation in healthy volunteers.

Simultaneous quantitation of levodopa and 3-O-methyldopa in human plasma by HPLC-ESI-MS/MS: application for a pharmacokinetic study with a levodopa/benserazide formulation.

A sensitive and simple method was developed for the quantitation of levodopa and its metabolite 3-O-methyldopa, in human plasma, after oral administration of tablet formulations containing levodopa (200 mg) and benserazide (50 mg). The analytes were extracted by a protein precipitation procedure, using carbidopa as an internal standard. A mobile phase consisting of 0.2% formic acid and acetonitrile (94:6, v/v) was used and chromatographic separation was achieved using ACE C(18) column (50 mm×4.6 mm i.d.; 5 µm particle size). Selected reaction monitoring was performed using the fragmentation transitions m/z 198→m/z 107, m/z 212→m/z 166 and m/z 227→m/z 181 for levodopa, 3-O-methyldopa and carbidopa, respectively. Calibration curves were constructed over the range 50.0-6000.0 ng/mL for levodopa and 25.0-4000.0 ng/mL for 3-O-methyldopa. The method shown to be specific, precise, accurate and provided recovery rates higher than 85% for all analytes. No matrix effect was detected in the samples. The validated method was applied in a pharmacokinetic study with a levodopa/benserazide tablet formulation in healthy volunteers.

Elevation of total homocysteine levels in patients with Parkinson's disease treated with duodenal levodopa/carbidopa gel.

Levodopa/carbidopa (LD/CD) application elevates total plasma homocysteine (thcys). We determined thcys-, LD- and 3-O-methyldopa (3-OMD) concentrations in 28 patients with Parkinson's disease (PD) on a LD/CD duodenal gel treatment. We found a distinct thcys increase (29.52 ± 28.98 µmol/l [median ± SD]) above the 15 µmol/l threshold and a significant (R = 0.7) correlation between LD and 3-OMD. thcys ascent was observed in relation with the onset of atherosclerosis, non-motor symptoms and polyneuropathy in PD patients in the long term.

Determination of L-dopa, carbidopa, 3-O-methyldopa and entacapone in human plasma by HPLC-ED.

The aim of the study was the development of analytical methods suitable for the quantification of L-dopa, carbidopa and entacapone in plasma of Parkinsonian patients treated with Stalevo(®). The metabolite 3-O-methyldopa was also determined to obtain some indications on the pharmacokinetics of L-dopa. For the simultaneous analysis of L-dopa, 3-O-methyldopa and carbidopa, a RP C18 column as the stationary phase and a mixture of methanol and a pH 2.88 phosphate buffer (8:92, v/v) as the mobile phase were used. A feasible plasma pre-treatment based on protein precipitation was implemented, obtaining extraction yield higher than 94% for all the analytes. For the analysis of entacapone a RP C8 column and a mixture of methanol, acetonitrile and a pH 1.90 phosphate buffer as the mobile phase (17.5:22.5:60, v/v/v) were used. A plasma pre-treatment procedure was developed, based on solid phase extraction of entacapone using Oasis HLB cartridges. Extraction yields were good, being always higher than 96%. Both methods, based on HPLC-ED (V=+0.8V), have been fully validated. Good linearity was obtained over the following concentration ranges: 100-4000 ng mL(-1) for L-dopa, 200-10,000 ng mL(-1) for 3-O-methyldopa, 25-4000 ng mL(-1) for carbidopa and 20-4000 ng mL(-1) for entacapone. Precision data were satisfactory, being R.S.D.% values lower than 5.64%; accuracy also resulted very good with recovery data higher than 90%. The proposed methods have been successfully applied to the analysis of patient plasma samples and seem to be suitable for therapeutic drug monitoring purposes.

Levodopa and 3-OMD levels in Parkinson patients treated with Duodopa.

We studied 19 patients (14 men, 5 women, Hoehn and Yahr (H&Y)> or =3) with advanced Parkinson's disease (PD) attending the Parkinson Institute, Milan, whose motor fluctuations and dyskinesia were not controlled by oral medications. After all oral PD medications had been withdrawn, they received a duodenal levodopa infusions (Duodopa, Solvay Pharmaceuticals) for 14h/day through a transabdominal port; levodopa boluses were administered in the morning and during "off" periods. The patients were evaluated by means of the UPDRS in the morning ("off") and 60-120min after the infusion ("on") at baseline and for a mean follow-up of 13.5+/-12.5months (up to 36months in 10 patients:). Levodopa (l-DOPA) and its metabolites were determined by HPLC with electrochemical detection. l-DOPA concentrations tended to higher in the afternoon (2008+/-345 vs 1713+/-274ng/mL) and correlated with the daily dose. O-methyldopa (OMD) levels correlated with l-DOPA levels, and the OMD/l-DOPA ratios were stable over the day. There was a relationship between decreasing UPDRS III scores and decreasing OMD/l-DOPA ratios. Dyskinesia (UPDRS IV, items 32-34) showed a clear improvement over time but there was no clear relationship with l-DOPA and OMD levels, or the OMD/l-DOPA ratio. The l-DOPA/dose ratio was stable over time, whereas OMD levels and the OMD/l-DOPA ratio decreased. It is conceivable that continuous infusion decreases metabolism possibly due to a reduction in methyl donor availability, as demonstrated by the increase in total homocysteine levels. Our results do not support the development of tolerance even after several months of continuous infusion, and indicate that pharmacodynamic factors play a role in afternoon off periods.

Quantitative analysis of levodopa, carbidopa and methyldopa in human plasma samples using HPLC-DAD combined with second-order calibration based on alternating trilinear decomposition algorithm.

An HPLC method combined with second-order calibration based on alternating trilinear decomposition (ATLD) algorithm has been developed for the quantitative analysis of levodopa (LVD), carbidopa (CBD) and methyldopa (MTD) in human plasma samples. Prior to the analysis of the analytes by ATLD algorithm, three time regions of chromatograms were selected purposely for each analyte to avoid serious collinearity. Although the spectra of these analytes were similar and interferents coeluted with the analytes studied in biological samples, good recoveries of the analytes could be obtained with HPLC-DAD coupled with second-order calibration based on ATLD algorithm, additional benefits are decreasing times of analysis and less solvent consumption. The average recoveries achieved from ATLD with the factor number of 3 (N=3) were 100.1+/-2.1, 96.8+/-1.7 and 104.2+/-2.6% for LVD, CBD and MTD, respectively. In addition, elliptical joint confidence region (EJCR) tests as well as figures of merit (FOM) were employed to evaluate the accuracy of the method.

Simultaneous determination of serum concentrations of levodopa, dopamine, 3-O-methyldopa and alpha-methyldopa by HPLC.

Levodopa is the medication of choice for Parkinson's disease. The biological complexity of levodopa and of its main derivatives makes their determination important in the clinical field. The aim of this study was to develop an HPLC method for the simultaneous determination of serum concentrations of levodopa, dopamine, 3-O-methyldopa and alpha-methyldopa. We compared UV and fluorimetric detection of native and derivatised compounds. Though less sensitive than other methods, UV detection is important to exclude naturally fluorescent, interfering substances. Fluorimetric detection of derivatised compounds is more sensitive than UV detection. Since 3-O-methyldopa does not react with the derivatising agent 1,2-diphenylethylenediamine, it cannot be detected. For simultaneous determination of the four compounds after pharmacological treatment of patients we therefore advise fluorimetric detection of the native compound.

Simultaneous kinetic-spectrophotometric determination of carbidopa, levodopa and methyldopa in the presence of citrate with the aid of multivariate calibration and artificial neural networks.

The kinetic methodology based on the difference of reaction rates, is based on the reaction between a common oxidizing agents such as tris(1,10-phenanthroline) and iron(III) complex (ferriin, [Fe (phen)3]3+) in the presence of citrate and spectrophotometrically, monitoring the changes of absorbance at the maximum wavelength of 511 nm. Experimental conditions such as pH, reagents and citrate concentrations were optimized, and the data obtained from the experiments were processed by several chemometric approaches, such as artificial neural network (ANN) and partial least squares (PLS). A set of synthetic mixtures of carbidopa (CD), levodopa (LD) and methyldopa (MD) was evaluated and the results obtained by the applications of these chemometric approaches were discussed and compared. It was found that the back propagation artificial neural network (BP-ANN) method afforded better precision relatively than those of radial basis function artificial neural networks (RBF-ANN) and PLS. The proposed method was also applied satisfactorily to the determination of carbidopa, levodopa and methyldopa in real samples.

Evidence for L-dopa incorporation into cell proteins in patients treated with levodopa.

Levodopa (L-dopa) is the most widely used agent for the symptomatic relief of Parkinson's disease. There is concern that chronic L-dopa treatment may be detrimental, with some studies suggesting that L-dopa may be neurotoxic. A potentially important mechanism whereby L-dopa may exert neurotoxic effects has been overlooked: that of the incorporation of L-dopa into proteins by protein synthesis. L-Dopa competes with tyrosine as a substrate in protein synthesis in vitro. We provide evidence that L-dopa can also be incorporated into proteins in vivo. Blood from L-dopa-treated and -non-treated patients was separated into protein, erythrocyte and lymphocyte fractions and levels of protein-incorporated dopa quantified by HPLC. Levels of protein-incorporated dopa were significantly increased in lymphocyte cell proteins from L-dopa-treated patients. This has not arisen from oxidative pathways as there was no evidence of oxidative damage to proteins. In addition, there was no increase in protein-incorporated dopa in erythrocytes, which are not actively synthesizing proteins. We suggest that protein-incorporated dopa could also be generated in the CNS. The accumulation of protein-incorporated dopa in cells is associated with oxidative stress and impaired function and could contribute to some of the problems associated with long-term L-dopa treatment.

A rapid high performance liquid chromatographic determination of methyldopa in human serum with fluorescence detection and alumina extraction: application to a bioequivalence study.

A simple and ultra rapid high performance liquid chromatographic (HPLC) method coupled with alumina extraction and fluorescence detection was described for determination of methyldopa in human serum. The drug and an internal standard were adsorbed onto alumina and eluted using acidic methanol. The eluate was directly injected onto ODS reverse phase column using a mixture of phosphate buffer (0.05 M) containing triethylamine (100 microl/l, v/v; pH 2.3) and methanol (92:8, v/v) at a flow rate of 2.1 ml/min as the mobile phase. The fluorescence detector excitation and emission wavelengths were set at 270 and 320 nm, respectively. No interference in the assay from any endogenous substances or other concurrently used drugs was observed and the retention times of I.S. and the drug were 1.7 and 2.4 min, respectively with total run time (injection to injection) of less than 3.5 min. The limit of quantification was evaluated to be 2 ng/ml. Validity of the method was studied and the method was precise and accurate with a linearity range from 20 ng/ml to 5000 ng/ml. This method has been used in a randomized crossover bioequivalence study of two different methyldopa preparations in 24 healthy volunteers.

Catechol-O-methyltransferase inhibition in erythrocytes and liver by BIA 3-202 (1-[3,4-dibydroxy-5-nitrophenyl]-2-phenyl-ethanone).

The present study evaluated the relationship between the degree of catechol-O-methyltransferase (COMT) inhibition in erythrocytes and liver by BIA 3-202 (1-[3,4-dihydroxy-5-nitrophenyl]-2-phenyl-ethanone) and determined its effects upon the O-methylation of L-DOPA in rats orally treated with L-DOPA plus benserazide. The soluble form of COMT (S-COMT) in erythrocytes was endowed with the same affinity as liver S-COMT for the substrate adrenaline. BIA 3-202 inhibited erythrocytes and liver S-COMT with ED50's of 1.9 (0.7, 3.1) and 1.9 (0.5, 3.2) (95% confidence limits) mg kg(-1), respectively. BIA 3-202 reduced the L-DOPA-induced rise of 3-O-methyl-L-DOPA in the peripheral circulation, striatal dialysate levels and striatum, and increased dopamine striatal levels. In BIA 3-202-treated rats the increase in L-DOPA in peripheral blood and striatal dialysates was significantly greater than in vehicle-treated rats. It is concluded that S-COMT activity in erythrocytes may provide important information on the pharmacodynamic profile of COMT inhibitors. The novel COMT inhibitor BIA 3-202 is a potent COMT inhibitor that enhances the availability of L-DOPA to the brain by reducing its O-methylation, which may prove beneficial in patients with Parkinson's disease treated with L-DOPA.

Quantification of methyldopa in human plasma by high-performance liquid chromatography-electrospray tandem mass spectrometry application to a bioequivalence study.

A method based on LC-MS-MS is described for the determination of methyldopa in human plasma using dopa-phenyl-D3 as the internal standard. The method has a chromatographic run time of 5.5 min and was linear in the range of 20-5000 ng/ml. The limit of quantitation was 20 ng/ml, the intra-day precisions were 7.3, 5.4 and 4.3% and the intra-day accuracies were -8.0, -1.3 and -2.0% for 30, 600 and 3000 ng/ml, respectively. The inter-day precisions were 7.7, 0.5 and 0.7% and the inter-day accuracies were 0.2, -1.1 and -2.3%, respectively, for the above concentrations. This method was employed in a bioequivalence study of two tablet formulations of methyldopa.

Pharmacokinetic study of methyldopa in aorta-coarctated rats using a microdialysis technique.

The pharmacokinetics of methyldopa (12.5, 25 and 50 mg kg(-1), i.p.) was studied in anesthetized sham-operated (SO) and abdominal aorta-coarctated (ACo) rats using a microdialysis technique. A non-linear relationship between the area under the curve (AUC) and dose was observed in SO rats. However, in ACo rats the AUC showed a proportional increase with dose. Abdominal aortic coarctation produced significant differences in the estimates of clearance (Cl) and the elimination rate constant from the dialysate (K(ed)) after the administration of 50 mg kg(-1)of methyldopa (K(ed)SO, 0.31 +/- 0.09; ACo, 0.66 +/- 0.09(*)h(-1): Cl SO, 30.8 +/- 10.1; ACo, 78.6 +/- 13.3(*)mlkg(-1)min(-1);n= 6,(*)P< 0.05 vs SO). In conclusion, this study, by using a microdialysis technique, suggests that abdominal aortic coarctation seems to produce changes in the pharmacokinetics of methyldopa in rats.

Study of the evolution of blood and striatal levels of methyldopa: a microdialysis study in sinaortic denervated rats.

Pharmacokinetic of methyldopa (50 mg kg(-1)i.p.) was studied in anesthetized sham operated and sinoaortic denervated (SAD) rats by using the microdialysis technique. Vascular shunt probe was inserted into the carotid artery and concentric probe was placed in the striatum. Levels of methyldopa were measured by HPLC-EC. The number of animals in each group was six and normal distribution of the variables of the study was assumed. Peak concentrations in arterial blood of methyldopa were similar in both groups of rats but the elimination rate constant was 0.31+/-0.09 h(-1)for sham rats (n =6) and 1.28+/-0.31 h(-1)for SAD rats (n =6, P<0.05). Low levels of methyldopa and a more pronounced decrease were seen in striatum of sinoaortic denervated rats. In conclusion, by using a microdialysis technique, different kinetic profiles of methyldopa were observed in sham operated and sinoaortic denervated rats.

Automated determination of levodopa and carbidopa in plasma by high-performance liquid chromatography-electrochemical detection using an on-line flow injection analysis sample pretreatment unit.

An automated analytical procedure is described for the parallel determination of L-3,4-dihydroxyphenylalanine (levodopa, L-dopa, LD) and the analogous hydrazine compound carbidopa (CD) in dog plasma by ion-pair high-performance liquid chromatography with electrochemical detection (HPLC-ED). After deproteinization of the plasma samples with perchloric acid the catecholamines were extracted from the supernatant by adsorption on a small column filled with alumina. The extraction and redissolution were automatically performed in a flow injection analysis unit (FIA) coupled to the HPLC system. The performance of the whole system was tested on dog plasma samples including specimens taken after oral administration of the anti-Parkinsonism drug Duellin, which is a combination tablet of levodopa and carbidopa.

Determination of alpha-methyldopa in human plasma by validated high-performance liquid chromatography with fluorescence detection.

A sensitive reversed-phase gradient elution high-performance liquid chromatographic method with fluorescence detection has been developed for the determination of alpha-methyldopa (AMD) in human plasma. Separation of the investigated compound and the 3,4-dihydroxyphenylalanine (DOPA) internal standard was achieved on a Nucleosil 7 C18 column with a 5 mM heptanesulphonic acid sodium salt containing 0.05 M potassium dihydrogenphosphate (pH 3.2)-acetonitrile mobile phase. The composition of the mobile phase was changed according to a linear gradient time program. Detection was performed at 270 nm fluorimetric excitation and 320 nm emission. The compounds were isolated from plasma by Bond-Elut C18 solid-phase extraction. The limit of quantitation was found to be 10 ng/ml plasma. The assay was validated with respect to accuracy, precision and system suitability. All validated parameters were found to be within the 20% required limits. On the basis of the sensitivity, linearity and validation parameters the developed analytical method was found to be suitable for application in a bioequivalency study.

Interaction of some drugs on the pharmacokinetics or pharmacodynamics of MPC-1304, a dihydropyridine Ca2+ antagonist.

The aim of this study was to assess the pharmacokinetics and subsequent pharmacodynamic interaction of MPC-1304, a dihydropyridine Ca2+ antagonist, with other drugs in animal experiments. We measured the systolic blood pressure and heart rate of conscious spontaneously hypertensive rats implanted with battery-operated biotelemetry devices after combined administration of various drugs. Cimetidine (10 mg/kg) did not affect the reduction in systolic blood pressure and the increase in heart rate induced by MPC-1304, whereas it significantly increased the plasma concentration of a metabolite of MPC-1304 (M-1) compared to that detected when MPC-1304 was administered alone. When MPC-1304 was consecutively administered in combination with rifampicin (400 mg/kg) for 9 days, the plasma concentrations of MPC-1304 and of M-1 significantly decreased compared to those found when MPC-1304 alone was given. In spite of these reductions in plasma concentrations, rifampicin did not attenuate the hypotensive action induced by MPC-1304. When prazosin, reserpine, or methyldopa was administered in combination with MPC-1304, the hypotensive action was enhanced as compared to that by MPC-1304 alone or to that by the co-administered drug used alone (prazosin, reserpine, or methyldopa). Quinidine (10 mg/kg) affected neither the hypotensive action induced by MPC-1304 nor the plasma concentrations of MPC-1304 and M-1. These results indicate that cimetidine and rifampicin interact with MPC-1304 pharmacokinetically, without apparently changing the hypotensive action of MPC-1304, whereas quinidine does not affect the metabolism of MPC-1304, and that other hypotensive drugs, such as prazosin, reserpine, and methyldopa, potentiate the hypotensive action of MPC-1304.