RAPID SPECTROPHOTOMETRIC DETERMINATION OF TRIFLUOPERAZINE DIHYDROCHLORIDE AS BASE FORM IN PHARMACEUTICAL FORMULATION THROUGH CHARGE-TRANSFER COMPLEXATION.

Two simple and selective spectrophotometric methods are described for the determination of trifluoperazine dihydrochloride (TFH) as base form (TFP) in bulk drug, and in tablets. The methods are based on the molecular charge-transfer complexation of trifluoperazine base (TFP) with either 2,4,6-trinitrophenol (picric acid; PA) or 2,4-dinitrophenol (DNP). The yellow colored radical anions formed are quantified at 410 run (PA method) or 415 nm (DNP method). The assay conditions were optimized for both the methods. Beer's law is obeyed over the concentration ranges of 1.5-24.0 pg/mL in PA method and 5.0-80.0 µg/mL in DNP method, with respective molar absorptivity values of 1.03 x 10(4) and 6.91 x 10(3) L mol-1 cm-1. The reaction stoichiometry in both methods was evaluated by Job's method of continuous variations and was found to be 1 : 2 (TFP : PA, TFP : DNP). The developed methods were successfully applied to the determination of TFP in pure form and commercial tablets with good accuracy and precision. Statistical comparison of the results was performed using Student's t-test and F-ratio at 95% confidence level and the results showed no significant difference between the reference and proposed methods with regard to accuracy and precision. Further, the accuracy and reliability of the methods were confirmed by recovery studies via standard addition technique.

Molecularly imprinted solid-phase extraction for selective trace analysis of trifluoperazine.

In this study, a novel sample clean-up technique based on the molecularly imprinted solid-phase extraction procedure is described for the determination of trifluoperazine (TFP) in biological fluids. The water-compatible molecularly imprinted polymers (MIPs) were prepared by using methacrylic acid as functional monomer, ethylene glycol dimethacrylate as cross-linker, chloroform as porogen and TFP as the template molecule. The novel imprinted polymer was used as a solid-phase extraction sorbent for the extraction of TFP from human serum and urine samples. Various parameters affecting the extraction efficiency of the polymer were evaluated. The selectivity of MIPs was evaluated by checking several substances with molecular structures similar to the template. The limits of detection and quantification for TFP in urine samples were 0.06 and 0.2 µg/L, respectively. These limits for TFP in serum samples were 0.15 and 0.4 µg/L, respectively. The recovery values for serum and urine samples were higher than 92 and 93%, respectively.

Biosensor for on-line fluorescent detection of trifluoroperazine based on genetically modified calmodulin.

This paper describes the development of a novel on-line biosensor based on a fluorescently labeled human calmodulin (CaM), hCaM M124C-mBBr, immobilized on controlled-pore glass (CPG), for the analysis of trifluoroperazine (TFP); a phenothiazine drug in human urine samples. The device was automated by packing hCaM M124C-mBBr-CPG in a continuous-flow microcell connected to a monitoring system, composed of a bifurcated optical fiber coupled to a spectrofluorometer. Operating parameters of the on-line biosensor (flow rate, sample injection volume, and carrier solution and buffer pH) were studied and optimized. Under the optimal conditions, the biosensor provides a detection and a quantification limit of 0.24 and 0.52 µg mL(-1), respectively, and a dynamic range from 0.52 to 61.05 µg mL(-1) TFP (n = 5, correlation coefficient 0.998). The response time (t(100)) was shorter than 42 s (recovery time <4.5 min) and reproducibility and repeatability of the TFP measurements, within the linear response range, were lower than 1.4 and 2.7%, respectively. The device was successfully applied to the analysis of TFP in spiked human urine samples with recoveries ranging between 97 and 101% and with RSDs lower than 5.9%.

Liquid chromatography-tandem mass spectrometry method for measurement of nicotine N-glucuronide: a marker for human UGT2B10 inhibition.

Nicotine is considered to be a specific substrate for UGT2B10, an isoform of human uridine diphosphate glucuronosyltransferase (UGT). In the present study, a sensitive and selective liquid chromatography/tandem mass spectrometry (LC-MS-MS) method for quantification of nicotine N-glucuronide in pooled human liver microsomal incubates was developed and validated. Proteins in a 200µL aliquot of incubation solution were precipitated by adding 40µL 35% perchloric acid. The overall extraction efficiency was greater than 98%. Nicotine N-glucuronide and internal standard were recorded using selected reaction monitoring in positive ion electrospray with ion transitions of m/z 339-163 and m/z 342-166, respectively. The linear calibration curve was obtained over the concentration range of 10-1000nM, with a lower limit of quantification of 10nM. The intra-day and inter-day precision (% CV) and accuracy (% bias) of the method were within 15% at all quality control levels. Nicotine glucuronide in processed samples was stable for 24h at room temperature and 48h at 4°C based on the stability experiments performed in this study. This established method was employed to evaluate the inhibitory effects of five target compounds including amitriptyline, hecogenin, imipramine, lamotrigine, and trifluoperazine on enzymatic activity of UGT2B10. IC(50) values for inhibition of nicotine N-glucuronidation by amitriptyline, imipramine, lamotrigine, and trifluoperazine were calculated. Trifluoperazine was found to be a non-substrate inhibitor for human UGT2B10.

Spectrophotometric determination of isopropamide iodide and trifluoperazine hydrochloride in presence of trifluoperazine oxidative degradate.

Four sensitive, selective and precise stability indicating methods for the determination of isopropamide iodide (ISO) and trifluoperazine hydrochloride (TPZ) in their binary mixture and in presence of trifluoperazine oxidative degradate (OXD). Method A is a derivative spectrophotometric one, where ISO was determined by first derivative (D(1)) at 226.4 nm while TPZ was determined by second derivative (D(2)) at 270.2 nm. Method B is the first derivative of the ratio spectra (DD(1)) spectrophotometric method, ISO can be determined by measuring the peak amplitude at 227.4 nm using 5 microg mL(-1) of OXD as a divisor, while TPZ can be determined by measuring the peak amplitude at 249.2 and 261.4 nm using 15 microg mL(-1) of ISO as a divisor. Method C is the isoabsorptive spectrophotometric method. This method allows determination of ISO and TPZ in their binary mixture by measuring total concentration of ISO and TPZ at their isoabsorptive point at lambda(229.8) nm (Aiso1) while TPZ concentration alone can be determined at lambda(max) 311.2 nm, then ISO concentration can be determined by subtraction. On the same basis TPZ can be determined in presence of ISO and OXD, where OXD concentration alone was determined by measuring the peak amplitude at lambda(281.6) and lambda(309.4) nm while total concentration of TPZ and OXD was determined at their isoabsorptive points at (Aiso2 = 270.2 nm), (Aiso3 = 310.6 nm) and (Aiso4 = 331.8 nm) then TPZ concentration was determined by subtraction. Method D is the multivariate calibration techniques [the classical least squares (CLS), principal component regression (PCR) and partial least squares (PLS)], using the information contained in the absorption spectra of ISO, TPZ and OXD mixtures. The selectivity of the proposed methods was checked using laboratory prepared mixtures. The proposed methods have been successfully applied to the analysis of ISO and TPZ in pharmaceutical dosage form without interference from other dosage form additives and the results were statistically compared with the reported method.

Potentiometric sensors for the determination of trifluoperazine hydrochloride in pharmaceutical preparations.

Trifluoperazine is widely used in the treatment of psychotic patients for its neuroleptic and antidepressive action. In this study, the construction, evaluation and application of new potentiometric sensors for trifluoperazine hydrochloride (TFPH) are described. The sensing membranes incorporated either ion-pair complexes of the trifluoperazine cation and phosphotungstic acid (PTA) or phosphomolybdic acid (PMA) or sodium tetraphenyl borate (NaTPB) as electroactive materials in poly(vinyl chloride) matrix membrane. The plasticizers used were di-n-butyl phthalate (DBPH) and tri-n-butyl phosphate (TBP). After a series of experiments, the best electrodes were based on PTA or PMA as electroactive materials and DBPH as plasticizer. A linear concentration range between 1 x 10(-5)-1 x 10(-2) M with a near Nernstian slope of 28.43 and 32.11 mV decade(-1), respectively, was obtained. The electrodes were characterized in terms of the composition, usable pH range, life span and response time. The selectivity coefficient values were calculated for different inorganic cations and sugars. Validation of the method shows the suitability of the electrodes for the determination of TFPH in pharmaceutical formulations.

Sensitive detection of trifluoperazine using a poly-ABSA/SWNTs film-modified glassy carbon electrode.

A poly-ABSA/SWNTs composite-modified electrode was fabricated by electropolymerizing aminobenzene sulphonic acid (ABSA) on the surface of glassy carbon electrode (GCE) modified with single-wall carbon nanotubes (SWNTs). SWNTs provide a 3D porous and conductive network for the polymer immobilization. The nanocomposite film was characterized by scanning electron microscope (SEM) and electrochemical impedance spectroscopy (EIS). The results indicated that this composite-modified electrode had strong electrocatalytic activity toward the oxidation of trifluoperazine (TFP). TFP could effectively accumulate on the modified electrode and generate a sensitive anodic peak at 0.72V (versus SCE) in pH 6.1 phosphate buffer solution. Under the selected conditions, the anodic peak current of TFP was linear with its concentration within the range from 1.0x10(-7) to 1.0x10(-5)molL(-1) and 1.0x10(-5) to 1.0x10(-4)molL(-1), and the detection limit was 1.0x10(-9)molL(-1) (S/N=3). This method was successfully applied to the detection of trifluoperazine in drug samples and the recovery was satisfactory. In comparison with the SWNTs/GCE or poly-ABSA/GCE prepared in the similar way, this composite-modified electrode exhibited better catalytic activity.

Quantitative prediction of in vivo inhibitory interactions involving glucuronidated drugs from in vitro data: the effect of fluconazole on zidovudine glucuronidation.

AIMS: Using the fluconazole-zidovudine (AZT) interaction as a model, to determine whether inhibition of UDP-glucuronosyltransferase (UGT) catalysed drug metabolism in vivo could be predicted quantitatively from in vitro kinetic data generated in the presence and absence bovine serum albumin (BSA). METHODS: Kinetic constants for AZT glucuronidation were generated using human liver microsomes (HLM) and recombinant UGT2B7, the principal enzyme responsible for AZT glucuronidation, as the enzyme sources with and without fluconazole. K(i) values were used to estimate the decrease in AZT clearance in vivo. RESULTS: Addition of BSA (2%) to incubations decreased the K(m) values for AZT glucuronidation by 85-90% for the HLM (923 +/- 357 to 91 +/- 9 microm) and UGT2B7 (478-70 microm) catalysed reactions, with little effect on V(max). Fluconazole, which was shown to be a selective inhibitor of UGT2B7, competitively inhibited AZT glucuronidation by HLM and UGT2B7. Like the K(m), BSA caused an 87% reduction in the K(i) for fluconazole inhibition of AZT glucuronidation by HLM (1133 +/- 403 to 145 +/- 36 microm) and UGT2B7 (529 to 73 microm). K(i) values determined for fluconazole using HLM and UGT2B7 in the presence (but not absence) of BSA predicted an interaction in vivo. The predicted magnitude of the interaction ranged from 41% to 217% of the reported AUC increase in patients, depending on the value of the in vivo fluconazole concentration employed in calculations. CONCLUSIONS: K(i) values determined under certain experimental conditions may quantitatively predict inhibition of UGT catalysed drug glucuronidation in vivo.

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.

Investigation into the applicability of the centrifugal microfluidics platform for the development of protein-ligand binding assays incorporating enhanced green fluorescent protein as a fluorescent reporter.

The incorporation of a protein-ligand binding assay into a centrifugal microfluidics platform is described. The platform itself is a disc-shaped polymer substrate, upon which a series of microfluidic channels and reservoirs have been machined. Centrifugal microfluidics platforms require no internal moving parts, and fluid propulsion is achieved solely through rotation of the disc. Fluid flow is controlled by passive valves, the opening of which is dependent on the angular frequency of the rotating platform, the channel dimensions, and the physical properties of the fluid. To evaluate the effectiveness of incorporating a protein-based assay onto the centrifugal microfluidics analytical platform, a class-selective, homogeneous assay for the detection of phenothiazine antidepressants was employed. This class of drugs is known to bind to calmodulin, a calcium binding protein. Specifically, a fusion protein between calmodulin and enhanced green fluorescent protein was utilized. Calmodulin undergoes a conformational change upon binding to phenothiazines that alters the fluorescence properties of the attached fluorescent protein, which can be correlated to the concentration of the drug present. Another important aspect of this work was to study the efficacy of the platform to perform reconstitution assays. To do this, the biological reagent was dried on the platform and rehydrated to carry out the assay. The ability to prealiquot reagents on the platform should enhance its versatility and portability. The integration of protein-based assays in this platform should be useful in the design of analytical systems for high-throughput screening of pharmaceuticals and clinical diagnostics.

Bead injection spectroscopy-flow injection analysis (BIS-FIA): an interesting tool applicable to pharmaceutical analysis. Determination of promethazine and trifluoperazine.

A bead injection spectroscopy-flow injection analysis (BIS-FIA) system for the spectrophotometric detection of promethazine and trifluoperazine is developed. The sensor is based in the oxidation of the phenothiazines by Fe(III) which is later determined by formation of the complex between Fe(II) and Ferrozine, [FeFz(3)](4-). Immediately, this complex is retained on a homogeneous bead suspension of Sephadex QAE A-25 resin (500 microl) which has been injected previously in the system to fill a commercial flow-cell (Hellma 138-OS). The use of BI with respect to the use of a reusable flow-through sensor is justified because the complex is so strongly retained on the beads that the regeneration of the solid support becomes extraordinarily difficult in the proposed method. At the end of the analysis, beads are automatically discarded from the flow-cell, by reversing the flow, and transported out of the system. The analytical signals are measured at a wavelength of 567 nm, corresponding to the absorbance of the complex. Using a sample volume of 600 microl, the analytical signal showed a very good linearity in the range 0.5-8.0 microgml(-1) and 0.5-10.0 microgml(-1), with detection limits of 0.09 and 0.14 microgml(-1) for promethazine and trifluoperazine, respectively. R.S.D.s (%) lower than 2% were obtained for both analytes. The proposed method is highly selective in the presence of other species that are normally encountered with these analytes. The sensor was satisfactorily applied to pharmaceutical preparations.

Colour reactions of PH. EUR. for identification of drugs using 1,3-dibromo-5,5-dimethylhydantoin (DBH) instead of elemental bromine. Analytical methods of pharmacopoeias with DBH in respect to environmental and economical concern, Part 10.

PH. EUR. 2002 and supplements identify aloes (Rosenthaler reaction), amiloride hydrochloride, chlorhexidine, dienestrol, quinidine sulphate, quinine hydrochloride and quinine sulphate (Thalleioquine reaction) and trifluoperazine hydrochloride using elemental bromine. This colour reaction can be performed better with 1,3-dibromo-5,5-dimethylhydantoin (DBH). Some prescriptions of PH. EUR. have been improved in respect to environmental and economical concern. The identification of amiloride hydrochloride with bromine water according to PH. EUR. 2002 or with DBH shows no UV fluorescence as reported in the pharmacopoeia.

Derivative spectrophotometric, thin layer chromatographic-densitometric and high performance liquid chromatographic determination of trifluoperazine hydrochloride in presence of its hydrogen peroxide induced-degradation product.

Three methods are presented for the determination of trifluoperazine HCl in presence of its hydrogen peroxide induced degradation product. The first method was based on measurement of first (1D) and second (2D) derivative amplitudes of trifluoperazine HCl in 0.1 N hydrochloric acid at the zero crossing point of its sulfoxide derivative, main degradation product, (at 268.4 and 262.5 nm for 1D and 2D, respectively). The second method was based on the separation of trifluoperazine HCl from its sulfoxide derivative followed by densitometric measurement of the intact drug spot at 255 nm. The separation was carried out on Merck aluminum sheet of silica gel 60 F(254), using chloroform-methanol (7:3 v/v) as mobile phase. The third method was based on high performance liquid chromatographic separation of trifluoperazine HCl from its sulfoxide derivative on reversed phase, ODS column, using a mobile phase of acetonitrile-phosphate buffer pH 4.2 (60:40 v/v) at ambient temperature. Quantitation was achieved with UV detection at 255 nm based on peak area. The first derivative spectrophotometric method was utilized to investigate the kinetics of the hydrogen peroxide degradation process at different temperatures. The apparent pseudo first-order rate constant, half life and activation energy were calculated.

[Determination of the mixed soporific by infrared subtractive spectroscopy technique].

A software of subtractive spectroscopy is provided on the NICOLET FTIR-560 infrared spectrometer. Some complicated processes of separation can be avoided by using the subtractive spectroscopy technique which simplifies the analytical procedure. An excellent infrared spectrum of a single component in a complex mixture is obtained successfully in this paper. By analysing and searching spectrum, the structures of the main components of the mixed soporific have been identified and it also improved the accuracy of appraising.

A fatality due to moclobemide.

A fatality due to ingestion of the antidepressant drug moclobemide is reported. Moclobemide is a selective and reversible inhibitor of monoamine oxidase type A. Previous reports have suggested that it is a safe drug even when taken in large quantities. The few reported fatalities have all been ascribed to serotonin syndrome, due to an interaction between moclobemide and other serotonergic agents. A 48-year-old woman with a history of depression and suicide attempts was found deceased at home. Autopsy revealed no evidence of significant natural disease or injury. Toxicologic analysis was performed and drug levels measured by capillary gas chromatography. The blood concentration of moclobemide was 137 mg/L and the liver concentration was 432 mg/kg. Low levels of diazepam, nordiazepam, and trifluoperazine were also detected. Death was considered to be due to acute poisoning by moclobemide. This case report is the first, to our knowledge, where death has been attributed to the toxic effects of moclobemide alone.

A systematic review and critical comparison of internal standards for the routine liquid chromatographic assay of amiodarone and desethylamiodarone.

Despite potential adverse effects, clinical use of amiodarone is increasing because of its efficacy in treating arrhythmias. Thus there is a continued need for a rapid, practical amiodarone assay to better study the relationship between serum concentrations and clinical effects and to guide safer dosing. Because the most widely used internal standard, L8040, is no longer available, a systematic comparison of potential alternatives was undertaken based on physicochemical and chromatographic characteristics. All amiodarone assays indexed on Medline were reviewed to produce a list of alternatives and five other potential substances considered based on previous experiences. An isocratic high-performance liquid chromatographic method was modified to allow simultaneous resolution of multiple compounds. The internal standard was expected to perform well in the solid-phase extraction of small sample volumes. No commercially available substances were able to duplicate all the advantages of L8040. Tamoxifen, the most acceptable alternative, was used to develop an assay to measure amiodarone and desethylamiodarone at concentrations as low as 0.25 mg/L in 100 microliters of serum (5 ng detected in a 20 microliters injection). Standard curves were linear over the range of concentrations found in our patients (0.25 to 8 mg/L), within-run coefficients of variation (CVs) averaged 5.3% for amiodarone and 2.9% for desethylamiodarone, and between-run CVs were 4.5% for amiodarone and 1.6% for desethylamiodarone.

Autoxidation of drugs: prediction of degradation impurities from results of reaction with radical chain initiators.

In the study of the degradation of drug substances by molecular oxygen, their specific reaction mechanisms must be taken into account. The rate-determining step is usually the reaction of the substrate with a radical chain initiator, which is often an unknown impurity. The reactivity and selectivity of autoxidation can be controlled better by using a radical chain initiator, such as AIBN, than by changing the temperature or the oxygen pressure. In this paper the products profiles of four pharmaceutical substances in a simple oxidation test with AIBN are compared with the results of long term natural stability tests or with already established stabilities.

Enzyme-amplified receptor assay screening test for chlorpromazine, trifluoperazine, and phencyclidine.

A new receptor based assay is described for the determination of classes of drugs which have high affinities for the acetylcholine receptor. The method is based upon the inhibition of the enzyme activity of an enzyme-drug conjugate by the binding to the receptor protein, and competition between free drugs and the enzyme-drug conjugate for a limited number of receptor sites. The activity of the enzyme marker system, glucose-6-phosphate dehydrogenase covalently conjugated to desipramine, is monitored by colorimetric detection of the rate of NADH formation at 340 nM. The procedure proposed is designed to provide a simple drug screen which can be done in a minimally equipped laboratory while achieving the required sensitivity. The technique is illustrated for three acetylcholine channel binding compounds: the hallucinogen phencyclidine (PCP) and the antipsychotic agents chlorpromazine and trifluoperazine. This procedure yields calibration curves with detection limits at nanomolar levels of drug, with binding responses dependent on the amounts of receptor and enzyme-labeled drug used. Aspecific binding responses of unlabeled enzyme to drug or receptor to compounds with low affinity for the receptor are shown to have minimal effect on the assay.