Analysis of perphenazine and fluphenazine by capillary electrophoresis coupled with tris (2,2'-bipyridyl) ruthenium (II) electrochemiluminescence detection.

The coupling of end-column tris (2,2'-bipyridyl) ruthenium (II) electrochemiluminescence (ECL) detection with capillary electrophoresis (CE) was developed for the analysis of two antipsychotic drugs, perphenazine (PPH) and fluphenazine (FPH). The parameters related to CE separation and ECL detection, including the detection potential, the buffer pH value and concentration, the separation voltage, and Ru(bpy)3(2+) concentration, were investigated in detail. Under optimum conditions, PPH and FPH were well separated and detected within 11 min. The linear ranges were 0.1-5 µM for PPH, and 0.1-7.5 µM for FPH, respectively. The limits of detection of PPH and FPH were 5 and 10 nM (S/N=3). The relative standard deviations (n=3) of the ECL intensity and the migration time were less than 2.5 and 0.65% in a day, and less than 3.4 and 1.7% in different three days. The proposed method was successfully applied to determine PPH and FPH in spiked urine samples with satisfactory results.

Ion beam analysis and PD-MS as new analytical tools for quality control of pharmaceuticals: comparative study from fluphenazine in solid dosage forms.

In order to evaluate the potential of accelerator based analytical techniques ((particle induced X-ray emission (PIXE), particle induced gamma-ray emission (PIGE), and particle desorption mass spectrometry (PD-MS)) for the analysis of commercial pharmaceutical products in their solid dosage form, the fluphenazine drug has been taken as a representative example. It is demonstrated that PIXE and PIGE are by far the best choice for quantification of the active ingredient (AI) (certification with 7% precision) from the reactions induced on its specific heteroatoms fluorine and sulfur using pellets made from original tablets. Since heteroatoms cannot be present in all types of drugs, the PD-MS technique, which makes easily the distinction between AI(s) and excipients, has been evaluated for the same material. It is shown that the quantification of AI is obtained via the detection of its protonated molecule. However, calibration curves have to be made from the secondary ion yield variations since matrix effects of various nature are characteristics of such mixtures of heterogeneous materials (including deposits from soluble components). From the analysis of solid tablets, (either transformed into pellets and even as received), it is strongly suggested that the physical state of the grains in the mixture is a crucial parameter in the ion emission and accordingly for the calibration curves. As a result of our specific (but not optimized) conditions the resulting precision is <17% with an almost linear range extending from 0.04 to 7.87 mg of AI in a tablet made under the manufacturer conditions (the commercial drug product is labeled at 5 mg).

Validation of derivative spectrophotometry method for determination of active ingredients from neuroleptics in pharmaceutical preparations.

First (DI) and second (D2) order derivative spectrophotometric method with an application of base line to peak technique was used for determination of active pharmaceutical ingredients (API) at two wavelengths: fluphenazine (D1 at lambda = 251 nm and lambda = 265 nm, D2 at lambda = 246 nm and lambda = 269 nm), pernazine (D1 at lambda = 246 nm and lambda = 258 nm, D2 at lambda = 254 nm and lambda = 262 nm), haloperidol (DI at = 235 nm and lambda = 253 nm, D2 at lambda = 230 nm and lambda = 246 nm), and promazine (D1 at lambda = 246 nm and lambda = 251 nm, D2 at lambda = 255 nm and lambda = 262 nm). Linear dependence of derivative values on analyte concentration is maintained in a range 3.12 microg x mL(-1) - 44.80 microg x mL(-1). Detection and determination limits are in the range 0.51 - 3.23 microg x mL(-1) and 1.27 microg x mL(-1) - 9.80 microg x mL(-1), respectively. Determination results of drug constituents are very accurate. Recovery percentage is in a range 95.50% - 103.60%.

Simultaneous chemiluminescence determination of promazine and fluphenazine using support vector regression.

In this work, chemiluminescence (CL) behaviors of two selected phenothiazines, namely promazine and fluphenazine hydrochloride, were investigated for their simultaneous determination using oxidation of Ru(bipy)(3)(2+) by Ce(4+) ions in acidic media. This method is based on the kinetic distinction of the CL reactions of fluphenazine and promazine with Ru(bipy)(3)(2+) and Ce(4+) system in a sulfuric acid medium. Least square support vector regression models were constructed for relating concentrations of both compounds to their CL profiles. The parameters of the model consisting of sigma(2) and gamma were optimized using all possible combinations of sigma(2) and gamma to select the model with the minimum root mean square cross validation. Under optimized conditions, the univariate calibration curve was linear over the concentration ranges of 0.4-30.0 microg mL(-1) and 0.07-5.0 microg mL(-1) with detection limits of 0.1 microg mL(-1) and 0.04 microg mL(-1) for promazine and fluphenazine, respectively. The influence of potential interfering substances on the determination of promazine and fluphenazine were studied. The proposed method was used for simultaneous determination of both compounds in synthetic mixtures and in spiked human plasma.

Second-derivative synchronous fluorescence spectroscopy for the simultaneous determination of fluphenazine hydrochloride and nortriptyline hydrochloride in pharmaceutical preparations.

A rapid, simple, and highly sensitive second-derivative synchronous fluorimetric (SDSF) method has been developed for the simultaneous analysis of binary mixtures of fluphenazine hydrochloride (FLZ) and nortriptyline hydrochloride (NTP) in their co-formulated tablets. The method is based upon measurement of the native fluorescence of these drugs at constant wavelength difference (Deltalambda) = 120 nm in acetic acid. The different experimental parameters affecting the fluorescence intensity of the studied drugs were carefully studied and optimized. The fluorescence-concentration plots were rectilinear over the range of 0.25-3.0 and 1-10 microg/ml for FLZ and NTP respectively, with lower detection limits (LOD) of 0.05 and 0.18 microg/ml and quantitation limits of 0.15 and 0.53 microg/ml for FLZ and NTP respectively. The proposed method was successfully applied for the determination of the studied compounds in their synthetic mixtures and in commercial co-formulated tablets. The results obtained were in good agreement with those obtained by the reference methods.

Spectrofluorometric determination of olanzapine and fluphenazine hydrochloride in pharmaceutical preparations and human plasma using eosin: application to stability studies.

A simple, rapid, and sensitive spectrofluorometric method has been developed for the determination of olanzapine (OLZ) and fluphenazine hydrochloride (FPZ HCI). The proposed method is based on the quantitative quenching effect of the studied drugs on the native fluorescence of eosin at pH 3.4 and 3.2 for OLZ and FPZ HCI, respectively. The fluorescence was measured at 547 nm after excitation at 323 nm. The fluorescence-concentration plots were rectilinear over the range of 0.05-1.0 and 0.10-1.0 microg/mL, with lower detection limits of 1.8 x 10(-3) and 1.2 x 10(-3) microg/mL, for OLZ and FPZ HCI, respectively. The proposed method was successfully applied to the analysis of commercial tablets and ampules containing the drugs, and the results were in good agreement with those obtained with reference methods. The proposed method was further applied to the determination of OLZ in spiked human plasma. The mean recovery was 98.62 +/- 0.24% (n = 4). The method was also used for stability studies of FPZ HCI upon oxidation with hydrogen peroxide, and the kinetics of the reaction were studied. A proposal for the reaction pathway was postulated.

Chemiluminescence sensor for the determination of perphenazine based on a molecular imprinted polymer.

A luminol-potassium ferricyanide-perphenazine CL reaction with high sensitivity for the determination of perphenazine was found. A perphenazine molecular imprinted polymer (MIP) was synthesized. Using the perphenazine MIP as a recognition material, a novel molecular imprinting-chemiluminescence (MI-CL) sensor for the determination of perphenazine was made based on the luminol-potassium ferricyanide-perphenazine CL reaction. The sensor displayed good selectivity and high sensitivity. The linear-response range of the sensor was 5.0 x 10(-8) - 1.0 x 10(-5) g/ml with a linear correlation coefficient of 0.9961. The detection limit was 2 x 10(-8) g/ml perphenazine and the relative standard deviation for 1.0 x 10(-6) g/ml perphenazine solution was 3.7% (n = 11). The sensor was applied to the determination of perphenazine in urine samples with satisfactory results.

Densitometric high performance thin-layer chromatography identification and quantitative analysis of psychotropic drugs.

A thin-layer chromatography (TLC)-densitometry method has been developed to identify and quantify haloperidol, amitriptyline, sulpiride, promazine, fluphenazine, doxepin, diazepam, trifluoperazine, clonazepam, and chlorpromazine in selected psychotropic drugs. Separation was performed on precoated silica gel 60 F254 TLC plates. Chromatograms were developed in various mobile phases, and 8 of 30 tested phases were selected based on spot location and developing time. The identification and quantification were carried out based on ultraviolet densitometric measurements at chosen wavelengths. In addition to retention coefficients, the absorption spectra recorded directly from chromatograms were also used in qualitative analysis. Under established experimental conditions, high sensitivity of the method was achieved. The limit of detection ranged from 0.009 to 0.260 microg, depending on the wavelength selected for measuring. A satisfactory recovery, ranging from 92.99 to 104.70%, was achieved for individual constituents.

Antimutagenic activity of new analogues of fluphenazine.

Fluphenazine (FPh) exhibited antimutagenic activity in lymphocyte cultures, markedly decreasing genotoxic effects of standard mutagenic agents present in cell cultures. However, the strong pharmacological activity of this neuroleptic drug, together with its serious side effects on the central nervous system, limits its use as an antimutagenic compound. In this paper we describe a route of chemical synthesis of FPh analogues that are more hydrophilic than the model compound, thus probably penetrate more weakly through the blood-brain barrier. These new analogues were tested for their antimutagenic and pro-apoptotic activities in human lymphocyte cultures, genotoxically damaged in vitro with benzo[a]pyrene [40 microM, 30 min] and subsequently cultured for 48 h in the presence of the tested compounds. The fluphenazine analogues enhanced apoptosis in genotoxically damaged lymphocytes more strongly than the model compound did. The increase of apoptotic cell frequency was the highest with compound 4a [2-(trifluoromethyl)-10-[3-(diethanolamino)-2-hydroxypropyl] phenothiazine]--a 35% higher effect than that of fluphenazine. The cytotoxicity of derivative 4a was the lowest among the tested compounds; it was 60% lower than that of fluphenazine. The antimutagenic effect of 4a was about 10% stronger than that of fluphenazine. Compound 4a also had the highest hydrophilicity of the new FPh analogues. Compound 4a was chosen for further study as a potentially usable antimutagen that would only weakly penetrate the central nervous system.

Late protective effects of the anticalmodulin drug fluphenazine on carbon tetrachloride-induced liver necrosis.

Fluphenazine (FP) treatment (50 mg/kg bw, ip in saline) 30 min before or 6 or 10 h after CCl4 administration (1 ml/kg ip in olive oil) significantly prevented the liver necrosis produced by the hepatotoxin at 24 h. FP had enhancing effects on the covalent binding of CCl4 reactive metabolites to cellular constituents and on CCl4 induced lipid peroxidation. FP lowered body temperature of the CCl4-poisoned animals during the 24 h observation period. The obtained results are compatible but do not prove the hypothesis that calmodulin (CaM) had participation in late occurring events preceding necrosis. FP lowering action on body temperature, however, might also play a role in the effects of this drug on the onset of CCl4 induced liver necrosis. FP levels in liver tissue as determined by gas chromatography-mass spectrometry evidenced the presence of the drug in amounts sufficient to inhibit CaM and that suggests that not all preventive effects of FP are due to its indirect actions on the central nervous system via decreased body temperature.

Radioimmunoassay for a new phenothiazine derivative and its application.

Fluphenazine-4-chlorophenoxy-isobutyrate ester, a new phenothiazine derivative was synthesized in the Institute for Drug Research Budapest. Radioimmunoassay was developed for the therapeutic monitoring of the drug level after intramuscular depot injection. The fluphenazine hapten was coupled to BSA by mixed-anhydride method. Antisera were produced to this conjugation in New-Zealand white rabbits and were tested for the antibody-titer. The specificity was tested by the cross-reaction with phenothiazine-analogues and other psychotropics. Strong cross-reaction was found with compounds possessing piperazine in side chain (trifluoperazine, perphenazine), but other psychotropic drugs did not react. Tritium-labelled trifluoperazine (spec. activity: 3.5 TBq/mmol) was used as a tracer in the radioimmunoassay. The detection limit was 75 pg with a CV of < 5% in 50 microliters plasma sample (equivalent to 1.5 ng/ml concentration) and a standard curve in the 3 ng/ml-50 ng/ml GYKI-22441 concentration range showed a CV of < 10%. Preliminary pharmacokinetic study was performed in Beagle dogs after intramuscular depot injection with GYKI-22441 in sesame oil in a dose of 0.1 mg/kg. The GYKI-22441 concentration of the plasma samples were measured by the RIA method during a 28-day interval after the treatment and was evaluated by the MultiCalc Immunoassay Data Management program (Pharmacia).

Evaluation of peroxyoxalate chemiluminescence postcolumn detection of fluphenazine in urine and blood plasma using high performance liquid chromatography.

Peroxyoxalate chemiluminescence may be used for sensitive postcolumn detection of phenothiazine analytes separated by high performance liquid chromatography with appropriate optimization of measurement conditions such as solvent, pH and oxalate ester. Detectability of fluorescent analytes by chemical excitation varies greatly, but analytes with low oxidation potentials are generally more readily detected at low levels, as demonstrated for phenothiazines, an important class of fluorescent drugs. Some improvement in detection limits is observed for fluphenazine when chemiluminescence detection is compared to conventional fluorescence detection. Because of the specificity of chemical excitation, fewer interferences from fluorescent impurities in a urine matrix are observed.

Noninvasive in vivo detection of a fluorinated neuroleptic in the human brain by 19F nuclear magnetic resonance spectroscopy.

A fluorinated long-acting neuroleptic (fluphenazine decanoate) was detected in the human brain by fluorine nuclear magnetic resonance spectroscopy (MRS). The MRS system used had a 3 Tesla magnetic field, corresponding to a frequency of 118 MHz for the fluorine nucleus (Bruker Medspec 3 Tesla, 60-cm magnet bore width). The spectra were obtained with a surface coil having a 10-cm diameter, which was centered alternatively on the frontal lobe and basal ganglia, and on the occipital lobe. The impregnation and elimination kinetics of fluphenazine were monitored during three sessions (4 hours, 5 days, and 11 days after injection of the compound). This technique can be usefully applied to the in vivo study of the pharmacokinetics and site of action of psychiatric compounds in man.

Development and application of a radioimmunoassay for fluphenazine based on monoclonal antibodies and its comparison with alternate assay methods.

The development of a monoclonal antibody towards fluphenazine allows the measurement of plasma concentrations of this highly potent neuroleptic. The method demonstrates sufficient sensitivity to measure 0.02 ng of fluphenazine per milliliter of plasma and employs a 150-microL plasma extract derived from a 2-mL plasma sample. The procedure is linear over the concentration range of 0.02 to 2.5 ng/mL, with a mean overall coefficient of variation of less than 3%. The validity of the described monoclonal-based RIA procedure was confirmed by comparison to alternate assay methods in replicate samples. Comparison to a newly developed HPLC-coulometric procedure in 159 samples showed a strong correlation, with a slope value of close to unity (1.0484) and a coefficient of correlation of 0.8136, while comparison to a previously developed polyclonal-based RIA procedure showed a correlation of 0.95 and a slope of 0.91 (n = 26).

[Stripping voltammetric determination of perphenazine and some antipsychotic drugs at carbon paste electrodes].

A sensitive electrochemical method for the determination of four drugs: perphenazine, fluphenazine chlorpromazine and trifluperazine is reported. These compounds exhibited accumulation at carbon paste electrodes in an open circuit. Differential pulse voltammetric determination gave linear calibration curves in the range of 0.02-1 microgram/ml. Detection limits found were 5-7 ng/ml. The drugs in tablets and body fluids were determined directly without separation. The preconcentration mechanism and determination conditions in a new kind of carbon paste piston electrode were investigated. Adsorption and extraction played important roles in accumulation process and the ratio of these two parts was measured by chronocoulometry.

Depot neuroleptic medication and serum levels by radioreceptor assay: prolactin concentration, electrocardiogram abnormalities and six-month outcome.

Twenty-six chronic schizophrenic patients on well-established depot neuroleptic regimes with stable doses (16 on fluphenazine decanoate, 10 on flupenthixol decanoate) had serum neuroleptic levels measured by radioreceptor assay (RRA) and were followed for six months. The serum prolactin (PRL) concentration and resting electrocardiogram (ECG) were also taken at the beginning of the study period. Correlations had previously been noted between RRA measured neuroleptic levels and outcome in both acute and chronic patients on oral medication. However, in this study of depot medication no significant correlations were found between serum neuroleptic concentration, serum prolactin concentration and the clinical state or outcome. The prevalence (33%) and type of ECG abnormality observed was similar to that previously reported.

Injection site leakage of depot neuroleptics: intramuscular versus subcutaneous injection.

In a cross-sectional study of injection site leakage of fluphenazine decanoate in 40 psychiatric outpatients, significantly more leakage occurred after intramuscular than after subcutaneous injection. Results of the study suggest that age, sex, body mass, and injection volume may also affect leakage.

Fluorinated psychopharmacological agents: noninvasive observation by fluorine-19 nuclear magnetic resonance.

Fluorinated psychopharmacological agents were measured with fluorine-19 nuclear magnetic resonance spectroscopy in the brain of intact rats that had been treated with fluphenazine. These in vivo experiments were compared to in vitro measurements of fluphenazine-treated rats. A high-field shift was observed in both in vivo and in vitro measurements. On the basis of the in vitro measurements, fluphenazine concentration in the brains of treated rats was estimated. Our observations demonstrate the feasibility of this technique for determining fluorinated neuroleptics in live mammals.

Liquid chromatographic determination of identity, content, and content uniformity of desipramine, fluphenazine, and promazine.

A liquid chromatographic procedure has been developed for the assay, content uniformity, and identification of single active ingredient formulations of desipramine, fluphenazine, and promazine. The drugs are extracted from formulations with methanol or dilute hydrochloric acid and quantitated against an internal standard (norephedrine). The drugs are identified by comparison of retention times with those of the reference standards.