Biosensor for the characterisation of hMAO B inhibitors and the quantification of selegiline.

A novel human monoamine oxidase B (hMAO B) based biosensor for inhibitory measurements was developed. It allows both the characterisation of the type of enzyme inhibition and the sensitive and simple determination of inhibitors like selegiline hydrochloride. The sensor consists of a screen printed carbon working electrode modified with 20% manganese dioxide (MnO2) and the enzyme hMAO B, which was immobilised on the electrode via a dialysis membrane (regenerated cellulose, molecular weight cut-off 14000). Inhibition of hMAO B is evaluated by adding different concentrations of the inhibitor selegiline hydrochloride to the enzyme and applying a defined amount of the hMAO B substrate phenylethylamine (PEA). The enzymatically formed H2O2 is amperometrically detected at 0.4V vs. Ag/AgCl in a flow injection analysis (FIA) system. With 100µM PEA the sensor showed a linear correlation between peak height and inhibitor concentration in a range of 0.51-3.25µg/mL selegiline hydrochloride. LOD and LOQ were determined to be 0.15 and 0.51µg/mL, respectively. The sensor showed a repeatability of 3.7% and an intermediate precision of 8.1%. The inhibition-based biosensor was successfully employed to quantify selegiline hydrochloride in pharmaceutical samples. Kinetic studies via Lineweaver-Burk plot and enzyme quantity vs. current plot revealed that the inhibition is irreversible.

Comparison of two methods for selegiline determination: A flow-injection chemiluminescence method using cadmium sulfide quantum dots and corona discharge ion mobility spectrometry.

Two analytical approaches including chemiluminescence (CL) and corona discharge ionization ion mobility spectrometry (CD-IMS) were developed for sensitive determination of selegiline (SG). We found that the CL intensity of the KMnO4-Na2S2O3 CL system was significantly enhanced in the presence of L-cysteine capped CdS quantum dots (QDs). A possible CL mechanism for this CL reaction is proposed. In the presence of SG, the enhanced CL system was inhibited. Based on this inhibition, a simple and sensitive flow-injection CL method was proposed for the determination of SG. Under optimum experimental conditions, the decreased CL intensity was proportional to SG concentration in the range of 0.01 to 30.0 mg L(-1). The detection limit (3σ) was 0.004 mg L(-1). Also, SG was determined using CD-IMS, and under optimum conditions of CD-IMS, calibration curves were linear in the range of 0.15 to 42.0 mg L(-1), with a detection limit (3σ) of 0.03 mg L(-1). The precision of the two methods was calculated by analyzing samples containing 5.0 mg L(-1) of SG (n=11). The relative standard deviations (RSDs%) of the flow-injection CL and CD-IMS methods are 2.17% and 3.83%, respectively. The proposed CL system exhibits a higher sensitivity and precision than the CD-IMS method for the determination of SG.

HPLC analysis of blood-brain barrier penetration of 4-fluorodeprenyl.

Validated HPLC analysis was developed in order to monitor the level of 4-fluorodeprenyl in rats. Male Wistar rats were intraperitoneally treated with 30 mg/kg of (-)-4-fluorodeprenyl. The rats were sacrificed after 5, 15, 30 and 60 min of treatment, and various tissues were isolated, such as serum, brain, CSF, liver, testis and lacrimal gland. Perchloric acid was given to aliquots, which were then homogenized, centrifuged and the supernatants were taken. The 4-fluorodeprenyl content was determined using reversed-phase HPLC, based on the comparison of the calibration line of the spiked samples. The level of 4-fluorodeprenyl was between 0.5 and 24 µg/g, showing maximum concentration in the brain and the liver after 5 min following its administration and in serum, CSF, testis, eyes and lacrimal gland after 15 min following its administration, while a relatively high concentration was found in the liver and the lacrimal gland.

Fabrication and in vitro characterization of polymeric nanoparticles for Parkinson's therapy: a novel approach

The objective of the research was to formulate and evaluate selegiline hydrochloride loaded chitosan nanoparticles for the Parkinson's therapy in order to improve its therapeutic effect and reducing dosing frequency. Taguchi method of design of experiments (L9 orthogonal array) was used to get optimized formulation. The selegiline hydrochloride loaded chitosan nanoparticles (SHPs) were prepared by ionic gelation of chitosan with tripolyphosphate anions (TPP) and tween 80 as surfactant. The SHPs had a mean size of (303.39 ± 2.01) nm, a zeta potential of +32.50mV, and entrapment efficiency of SHPs was 86.200 ± 1.38%. The in vitro drug release of SHPs was evaluated in phosphate buffer saline (pH 5.5) using goat nasal mucosa and found to be 82.529% ± 1.308 up to 28 h. Release kinetics studies showed that the release of drug from nanoparticles was anomalous (non-fickian) diffusion indicating the drug release is controlled by more than one process i.e. superposition of both phenomenon, the diffusion controlled as well as swelling controlled release. SHPs showed good stability results as found during stability studies at different temperatures as mentioned in ICH guidelines. The results revealed that selegiline hydrochloride loaded chitosan nanoparticles are most suitable mode of delivery of drug for promising therapeutic action.

O objetivo da pesquisa foi formular e avaliar nanopartículas de quitosana contendo cloridrato de selegilina para terapia do Parkinson, a fim de melhorar o seu efeito terapêutico e reduzir a frequência de dosagem. Método de Taguchi, de planejamento experimental, (L9 matriz ortogonal) foi usado para obter a formulação otimizada. As nanopartículas de quitosana contendo cloridrato de selegilina (PCHs) foram preparadas por gelificação iônica de quitosana com ânions tripolifosfato (TPP) e Tween 80 como tensoativo. As PCHs apresentaram tamanho médio de (303.39 ± 2,01) nm, potencial zeta de +32.50 mV e eficiência de encapsulação de 86.200±1,38%. A liberação do fármaco in vitro foi avaliada em solução salina de tampão fosfato (pH 5,5), usando a mucosa nasal de cabra e o resultado encontrado foi de 82.529% ± 1.308, acima de 28 h. Estudos de cinética de liberação mostraram que a liberação do fármaco das nanopartículas foi por difusão anômala (não fickiana), indicando que é controlada por mais de um processo, ou seja, a superposição dos fenômenos de difusão controlada e intumescimento. As PCHs mostraram resultados de boa estabilidade, encontrada durante os estudos de estabilidade em temperaturas diferentes, como mencionado em diretrizes do ICH. Os resultados revelaram que o sistema de nanopartículas de quitosana contendo cloridrato de selegilina é o mais adequado sistema de liberação de fármacos de ação terapêutica promissora.

Optimization and validation of a dissolution test for selegiline hydrochloride tablets by a novel rapid HPLC assay using a monolithic stationary phase.

The present study reports the optimization and validation of a dissolution test for selegiline.HCl tablets using a new high-performance liquid chromatographic (HPLC) method. Rapid separation of the analyte from sample matrix was achieved in less than 60s using a Cromolith RP-18e monolithic column using UV detection at 220 nm. Thorough validation of the assay based on pre-defined criteria included linearity, LOD/LOQ, accuracy, precision, selectivity and ruggedness. The dissolution test was optimized in terms of dissolution medium, basket (type I)/paddle (type II) agitation and rotation speed. Its ruggedness was also validated. The presented analytical and dissolution procedures are currently being applied in the quality and stability control of Cosmopril tablets (5mg/tablet selegiline.HCl, Cosmopharm Ltd., Korinthos, Greece).

Parallel analysis of stimulants in saliva and urine by gas chromatography/mass spectrometry: perspectives for "in competition" anti-doping analysis.

Stimulants are banned by the World Anti-Doping Agency (WADA) if used "in competition". Being the analysis of stimulants presently carried out on urine samples only, it might be useful, for a better interpretation of analytical data, to discriminate between an early intake of the substance and an administration specifically aimed to improve the sport performance. The purpose of the study was to investigate the differences, in terms of excretion/disappearance of drugs, between urine and oral fluid, a sample that can reflect plasmatic concentrations. Oral fluid and urine samples were collected following oral administration of the following stimulants: modafinil (100 mg), selegiline (10 mg), crotetamide/cropropamide (50 mg each), pentetrazol (100 mg), ephedrine (12 mg), sibutramine (10 mg), mate de coca (a dose containing about 3mg of cocaine); analysis of drugs/metabolites was carried out by gas chromatography/mass spectrometry (GC/MS) in both body fluids. Our results show that both the absolute concentrations and their variation as a function of time, in urine and in oral fluid, are generally markedly different, being the drugs eliminated from urine much more slowly than from oral fluid. Our results also suggest that the analysis of oral fluid could be used to successfully complement the data obtained from urine for "in competition" anti-doping tests; in all those cases in which the metabolite(s) concentration of a substance in urine is very low and the parent compound is not detected, it is indeed impossible, relying on urinary data only, to discriminate between recent administrations of small doses and remote administrations of higher doses.

Separation of enantiomers of deprenyl with various CDs in CE and the effect of enantiomer migration order on enantiomeric impurity determination of selegiline in active ingredients and tablets.

Opposite affinity pattern of enantiomers of the antiparkinsonian chiral drug deprenyl (DEP) was observed towards various neutral and charged derivatives of -CD. The effect of the enantiomer migration order on the LOD of enantiomeric impurity of R-DEP (selegiline) was studied for the standard substances and in the tablets from three different suppliers. The influence of injection mode on the LOD of a minor enantiomeric impurity was also studied and the CE method was compared with the pharmacopoeial HPLC method using a commercially available chiral column Chiralcel OD-H. The optimized CE method was more suitable for low-level enantiomeric impurity determination in selegiline compared to the pharmacopoeial HPLC method.

Simultaneous determination of selegiline and desmethylselegiline in human body fluids by headspace solid-phase microextraction and gas chromatography-mass spectrometry.

A method for the simultaneous determination of selegiline and its metabolite, desmethylselegiline, in human whole blood and urine is presented. The method, which combines a fiber-based headspace solid-phase microextraction (SPME) technique with gas chromatography-mass spectrometry (GC-MS), required optimization of various parameters (e.g., salt additives, extraction temperatures, extraction times and the extraction properties of the SPME fiber coatings). Pargyline was used as the internal standard. Extraction efficiencies for both selegiline and desmethylselegiline were 2.0-3.4% for whole blood, and 8.0-13.2% for urine. The regression equations for selegiline and desmethylselegiline extracted from whole blood were linear (r(2)=0.996 and 0.995) within the concentration ranges 0.1-10 and 0.2-20 ng/ml, respectively. For urine, the regression equations for selegiline and desmethylselegiline were linear (r(2)=0.999 and 0.998) within the concentration ranges 0.05-5.0 and 0.1-10 ng/ml, respectively. The limit of detection for selegiline and desmethylselegiline was 0.01-0.05 ng/ml for both samples. The lower and upper limits of quantification for each compound were 0.05-0.2 and 5-20 ng/ml, respectively. Intra- and inter-day coefficients of variation for selegiline and desmethylselegiline in both samples were not greater than 8.7 and 11.7%, respectively. The determination of selegiline and desmethylselegiline concentrations in Parkinson's disease patients undergoing continuous selegiline treatment is presented and is shown to validate the present methodology.

High-performance liquid chromatographic-mass spectrometric determination of methamphetamine and amphetamine enantiomers, desmethylselegiline and selegiline, in hair samples of long-term methamphetamine abusers or selegiline users.

We devised a highly sensitive method for simultaneously determining methamphetamine (MA) and amphetamine (AP) enantiomers, desmethylselegiline (DMSG) and selegiline (SG), in human hair using a derivatization technique and high-performance liquid chromatography-electrospray ionization-mass spectrometry (HPLC-ESI-MS). MA and AP enantiomers and DMSG were effectively converted to trifluoroacetic acid (TFA) derivatives, and the sensitivity of MA and DMSG increased five times over compared with that of free bases. The TFA derivatives of each compound were stable within one week in a stock solution of methanol or for 24 h in the HPLC mobile phase (mixture of methanol and ammonium formate buffer). Each compound was well separated, and calibration curves were linear in the concentration range 0.04-40 ng/mg for MA enantiomers, SG and DMSG, and 0.2-40 ng/mg for AP enantiomers. The accuracy and precision of the method were evaluated, and relative standard deviations were within 7%. Our method was successfully applied to hair samples obtained from long-term MA abusers and SG users. (+)-MA and (+)-AP were detected from three MA abusers at concentrations of 0.79-20.85 and 0.04-3.30 ng/mg, respectively. On the other hand, (-)-MA, (-)-AP, DMSG, and SG were detected in three SG users at concentrations of 2.48-9.05, 0.72-3.10, 0.12-0.59, and 0-0.04 ng/mg, respectively. Based on our obtained data, discrimination of MA abusers from SG users was considered to be possible by comparing optical isomers of MA and AP, the existence of DMSG and/or SG, and the concentration ratio of AP to MA in hair samples.

The use of LC/MS, GC/MS, and LC/NMR hyphenated techniques to identify a drug degradation product in pharmaceutical development.

Understanding drug degradation in the formulated product is critical in pharmaceutical development as it has significant impacts on drug efficacy, safety profile and storage conditions. As a result, identification of degradation compounds has taken an important role in the drug development process. In this study, various hyphenated analytical techniques, such as liquid chromatography mass spectrometry (LC/MS), gas chromatography mass spectrometry (GC/MS), and liquid chromatography nuclear magnetic resonance with a solid phase extraction interface (LC/SPE/NMR), have been applied to the identification of a drug degradation product which grew over time in the stability study of the drug product. The target unknown is less polar and more unsaturated than the drug substance based upon reverse phase HPLC relative retention time and UV spectra. It is not ionizable by electrospray ionization (ESI) or atmospheric pressure chemical ionization (APCI) in either a positive or a negative mode. The unknown was isolated by an HPLC fraction collector and enriched by solid phase extraction. GC/MS with chemical ionization (CI) was employed to determine the molecular weight of this compound. Its fragmentation pattern was determined by CI-MS/MS using an ion trap mass spectrometer. The isolated material was also analyzed by LC/SPE/NMR, from which the structure of this compound was further characterized. The study utilizes a combination of various hyphenated analytical techniques to obtain complimentary information for structure elucidation of the unknown. The combination approach is critical for unambiguous impurity structure elucidation in drug degradation studies of pharmaceutical drug products.

A rapid, sensitive electron-capture gas chromatographic procedure for analysis of metabolites of N-methyl,N-propargylphenylethylamine, a potential neuroprotective agent.

INTRODUCTION: N-Methyl,N-propargyphenylethylamine (MPPE) is a novel analog of (-)-deprenyl, a drug prescribed for Parkinson's disease and shown to have neuroprotective and neurorescue properties in a wide variety of in vitro and in vivo models. MPPE is also neuroprotective, but has the advantage over (-)-deprenyl of not being metabolized to amphetamine or N-methylamphetamine. METHOD: In this paper, extractive derivatization with pentafluorobenzenesulfonyl chloride (PFBSC) followed by electron-capture gas chromatography was utilized to study the metabolism of MPPE. RESULTS: The procedure is rapid and reproducible, giving derivatives with excellent chromatographic properties. Using this procedure, it has now been shown that beta-phenylethylamine (PEA), N-methylphenylethylamine (N-methylPEA) and N-propargylphenylethylamine (N-propargylPEA) are formed from MPPE during incubation of this drug with human liver microsomes. Levels of all three metabolites were shown to increase with increasing time of incubation with the microsomes. DISCUSSION: Extractive derivatization with PFBSC followed by electron-capture gas chromatography represents an efficient means of separating and quantitating the metabolites of MPPE, a novel neuroprotective agent.

In vitro formation of selegiline-N-oxide as a metabolite of selegiline in human, hamster, mouse, rat, guinea-pig, rabbit and dog.

It is well established in the litrature, that selegiline is metabolised to its N-dealkylated metabolites, N-desmethylselegiline, methamphetamine and amphetamine. However, most studies on selegiline metabolism did not characterize the species differences in the formation of the metabolites. Therefore, in this study, we investigated the in vitro metabolism of selegiline in liver microsomes of different species. In addition, to the previously well-characterized metabolites, selegiline-N-oxide (selegiline-NO) was found to be formed as a metabolite of selegiline in rat liver microsomal preparation. The results of experiments with liver microsomes from other species indicated species differences in the rate and extent of formation of selegiline-NO. The dog and hamster liver microsomal preparations were the most active in terms of selegiline-NO production, whereas little selegiline was metabolized to its N-oxide in human liver microsomes. When selegiline-NO was incubated with rat liver microsomes, no metabolism occurred. When a short incubation time was applied in selegiline expriments no increase in the amount of selegiline-NO was detected. Accordingly, it was clear that selegiline was not metabolized to the N-dealkylated or N,N-bis-dealkylated compounds via selegiline-NO. Studies with different isoenzyme inhibitors indicated that the formation of selegiline-NO might be catalyzed at least partly by cytochrome P450 (CYP) 2D6 and CYP3A4. With the exception of hamster microsomes in the microsomal preparations in vitro, the formation of the R,S-stereoisomer of selegiline-NO was preferred.

Incorporation of selegiline metabolites into hair after oral selegiline intake.

We have previously shown that melanin in human hair has a great impact on the incorporation of codeine into hair. The present study on 10 subjects was performed to investigate whether or not these findings could also be extrapolated to other therapeutic drugs. We chose selegiline because it metabolizes to two commonly abused central stimulants, methamphetamine and amphetamine. The results would therefore also be of interest when studying the intake of such drugs and their incorporation into human hair. Selegiline and metabolites were determined by gas chromatography-mass spectrometry, total melanin by spectrophotometry, and pyrrole-tricarboxylic acid by high-performance liquid chromatography with ultraviolet detection. Our results show strong positive exponential relationships (y = e(x)) between melanin and the metabolites, which for methamphetamine improved by normalizing for plasma area under the curve. We conclude that the major metabolites of selegiline can be detected in hair up to four weeks after a single oral dose and that the incorporation closely relates to the melanin contents.

Analysis of deprenyl metabolites in the rat brain using HPLC-ES-MS.

Methylamphetamine and amphetamine, the two major metabolites of deprenyl in the rat brain were analyzed using HPLC method combined with electrospray-mass spectrometer. (-)-Deprenyl and (+)-deprenyl were orally administered to rats either in a single dose of 10 mg/kg, or three times a week for three weeks. The metabolites were determined in four different parts of the rat brain, such as in the frontal cortex, corpus striatum, hippocampus, and hypophysis. The ratio of methylamphetamine to amphetamine was also compared after (-)-deprenyl and (+)-deprenyl treatments.

Stereochemical determination of selegiline metabolites in postmortem biological specimens.

In this study, findings related to an aircraft accident are reported. Biological specimens collected at autopsy from the pilot of the fatal accident and two types of tablets found at the accident scene were submitted for toxicological evaluation. It was determined that the pilot was dead at the crash site and the cause of death was multiple traumatic injuries. The tablets were identified as selegiline and levodopa, commonly prescribed for the treatment of Parkinson's disease. Selegiline, a stereospecific compound, is biotransformed into (-)-N-desmethylselegiline, (-)-methamphetamine, and (-)-amphetamine. The latter two levorotatory metabolites cannot be easily distinguished by routine analysis from their dextrorotatory isomers, which are controlled substances. It was, therefore, prudent to differentiate these isomers to determine if they resulted from the ingestion of a controlled substance, (+)-methamphetamine. Initial immunoassay drug screenings revealed the presence of amphetamine class drugs (867 ng/mL) in urine, amphetamine/methamphetamine (261 ng/mL) in urine, and methamphetamine (46 ng/mL) in blood. The gas chromatography-mass spectrometry (GC/MS) results revealed the presence of methamphetamine in the concentrations of 76 ng/mL of blood and 685 ng/mL of urine. The concentration of amphetamine was 52 ng/mL in blood and 320 ng/mL in urine. To determine the stereospecificity of these amines, the isolated amines from the biosamples were derivatized by a stereospecific agent, (S)-(-)-N-(trifluoroacetyl)-prolyl chloride, and characterized by a GC/MS method to be levorotatory. The 2.14 ratio of (-)-methamphetamine to (-)-amphetamine concentrations in the urine was consistent with a selegiline study in the recent literature. The stereospecific analysis, in conjunction with the history of the pilot being on Parkinson's medications, suggests that the source of these amines was selegiline. This conclusion substantiates the importance of the identification of enantiomers in evaluating and interpreting related analytical results for accident investigations.

Metabolism of (-)-deprenyl and PF-(-)-deprenyl in brain after central and peripheral administration.

We examined the cerebral metabolism of L-deprenyl and its fluoro-derivative pF-deprenyl, assaying the parent compounds, their metabolites desmethyl deprenyl, L-amphetamine, and L-methamphetamine, and the fluoro analogs of these metabolites. We compared the levels of the metabolites after subcutaneous injection with those after intracerebral administration (via microdialysis) of the parent compounds. The assay of the parent compounds and their metabolites was by GC-MS measurement of the components of brain microdialysate samples. After their subcutaneous administration, deprenyl and F-deprenyl rapidly entered the brain and then their concentration decreased, with an approximate half-life of 4.5 h. After the intracerebral administration the diffusion from the site of administration was minor. A small fraction (a few percent) of the intracerebrally administered deprenyl was metabolized in situ in the brain possibly by a nonenzymatic process. Metabolism of pF-deprenyl was somewhat more rapid. The higher cerebral levels of metabolites after the subcutaneous administration indicate their exogenous origin-metabolism of parent compounds in the periphery and penetration of the brain by the metabolites.

Hair analysis for drugs of abuse. IX. Comparison of deprenyl use and methamphetamine use by hair analysis.

Deprenyl (DPN) and its metabolites, desmethyl deprenyl (desmethyl DPN), methamphetamine (MA) and amphetamine (AP), in the hair of rats and humans dosed with DPN were analyzed by selected ion monitoring of gas chromatograph-mass spectrometry (GC-MS-SIM). After intraperitoneal administration of DPN-HCl to rats (10 mg/kg/d, 10d, n = 3), the area under the concentration versus time curves (AUCs) of DPN and its metabolites in the rat plasma were compared with the concentrations of drugs in rat hair newly grown for 4 weeks. The concentrations of DPN, desmethyl DPN, MA and AP in the rat hair were 0.97 +/- 0.06, 0.68 +/- 0.02, 14.04 +/- 0.54 and 13.20 +/- 1.09 ng/mg, and the ratios of the concentrations in the hair to AUCs in their plasma were 0.05: 0.02: 0.3: 0.2, respectively. This fact suggested that the incorporation rates of DPN and desmethyl DPN into hair from blood were relatively lower than those of MA and AP. The method was applied to determination of the metabolites in scalp hair, beard and urine of humans who orally ingested DPN (15 mg/d, 5 d, n = 3). DPN (trace level), desmethyl DPN (0.17-0.29 ng/mg), MA (1.30-2.25 ng/mg) and AP (0.42-0.99 ng/mg) were detected in the scalp hair collected three weeks after the first intake, while in beard or urine these drugs were detected only for a few days.(ABSTRACT TRUNCATED AT 250 WORDS)

[Survey of the analysis of selegiline]. / Attekintés a selegilin hatóanyag analitikájáról.

The characteristics of the selegiline active substance, identification tests, purity tests and the determinations of the active substance content are summarized in the paper. Fast, simple, selective reverse phase HPLC method is used for purity tests and active substance determinations. The validation of the determination of the active substance and the purity test is presented. The solvent residues were determined in vapour phase by the headspace method. A potentiometric acidi-alkalimetric titration, carried out in nonaqueous solution with the application of a titriprocessor is applied as an assay. A thermoanalytical method developed for the purity determinations is also published in this issue by Ladányi et al.

Chromatographic studies on the binding, action and metabolism of (-)-deprenyl.

Serum binding, the effect on striatal dopamine release and the metabolism of (-)-deprenyl [N-methyl-N-propargyl(2-phenyl-1-methyl)ethylammonium chloride], TZ-650 [N-methyl-N-propargyl(2-phenyl)ethylammonium chloride] and J-508 [N-methyl-N-propargyl(indanyl)ammonium chloride] were investigated using various chromatographic methods. A strong interaction between (-)-deprenyl and macroglobulins was found. Deprenyl enhanced the dopamine release from striatal slices of the rat brain and also inhibited the dopamine-DOPAC conversion. Deprenyl analogues showed either smaller or no effect. Hydroxylation of (-)-deprenyl takes place in the para position, in addition to the usual oxidative N-dealkylations, which are known from various metabolic studies on N-substituted phenylalkylamines.