Cytotoxicity evaluation of haloperidol, clozapine and a new molecule with antipsychotic potential, PT-31, in NIH-3T3 cells

Abstract Schizophrenia is an illness that affects 26 million people worldwide. However, conventional antipsychotics present side effects and toxicity, highlighting the need for new antipsychotics. We aimed to evaluate the cytotoxicity of haloperidol (HAL), clozapine (CLO), and a new molecule with antipsychotic potential, PT-31, in NIH-3T3 cells. The neutral red uptake assay and the MTT assay were performed to evaluate cell viability and mitochondrial activity, morphological changes were assessed, and intracellular reactive oxygen species (ROS) detection was performed. HAL and CLO (0.1 µM) showed a decrease in cell viability in the neutral red uptake assay and in the MTT assay. In addition, cell detachment, content decrease, rounding and cell death were also observed at 0.1 µM for both antipsychotics. An increase in ROS was observed for HAL (0.001, 0.01 and 1 µM) and CLO (0.01 and 1 µM). PT-31 did not alter cell viability in any of the assays, although it increased ROS at 0.01 and 1 µM. HAL and CLO present cytotoxicity at 0.1 µM, possibly through apoptosis and necrosis. In contrast, PT-31 does not present cytotoxicity to NIH-3T3 cells. Further studies must be performed for a better understanding of these mechanisms and the potential risk of conventional antipsychotics

Hollow Fiber Liquid-Phase Microextraction At-Line Coupled to Capillary Electrophoresis for Direct Analysis of Human Body Fluids.

A simple and cheap all-in-one concept for at-line coupling of hollow fiber liquid-phase microextraction (HF-LPME) to commercial capillary electrophoresis (CE) is demonstrated, which enables the direct analysis of complex samples. A disposable microextraction device compatible with injection systems of Agilent CE instruments is proposed, which consists of a short segment of a porous HF attached to a tapered polypropylene holder. The holder maintains a constant position of the HF in a CE vial during extraction and simultaneously guides the injection end of a separation capillary into the HF lumen for automated CE injection and analysis. In a typical analytical procedure, the HF is impregnated with a water-immiscible solvent, its lumen is filled with 5 µL of an aqueous acceptor solution, and the microextraction device is placed in a 2 mL glass CE vial containing 550 µL of a donor solution. The vial is agitated at 750 rpm for 10 min, and the resulting acceptor solution is injected directly from the HF lumen into the commercial CE. No additional manual handling is required, except for the transfer of the CE vial to the CE autosampler. Multiple complex samples can be simultaneously pretreated in a multiple-well plate format, thus significantly reducing the total analysis time. Suitability of the analytical method is demonstrated by the direct determination of model basic drugs (nortriptyline, haloperidol, loperamide, and papaverine) in physiological solutions, urine, and dried blood spot (DBS) samples. Repeatability of the method is better than 12.8% (%RSD), extraction recoveries range between 34 and 76%, and enrichment factors are 37-84. The method is linear in a range of 2 orders of magnitude (R2 ≥ 0.9977) with limits of detection of 0.7-1.55 µg/L. The method has a high potential for the direct analysis of DBS samples since DBS elution and HF-LPME are performed simultaneously during the 10 min agitation. The manual DBS handling is thus reduced to inserting the DBS punch into the CE vial only. Moreover, the universal character of the HF-LPME might extend the applicability of the method to a wide range of analytes/matrices, and combination with other commercial detectors might improve the selectivity/sensitivity of the CE analysis.

Repeated attempted homicide by administration of drugs documented by hair analysis.

Attempted murder by repeated poisoning is quite rare. The authors describe the case of a 62-year-old man who was admitted to an intensive care unit (ICU) for neurological disturbances complicated by inhalation pneumopathy. He presented a loss of consciousness while his wife was visiting him at the ICU (H0). Forty-eight hours later (H48), police officers apprehended the patient's wife pouring a liquid into his fruit salad at the hospital. Toxicological analyses of a blood sample and the infusion equipment (H0), as well as the fruit salad and its container (H48), confirmed the attempted poisoning with cyamemazine (H0) and hydrochloric acid (H48). In order to evaluate the anteriority of poisonings, hair analysis was requested and the medical records of the 6 previous months were also examined. Two 6-cm brown hair strands were sampled and the victim's medical record was seized in order to determine the treatments he had been given during the previous six months. Segmental hair testing on two 6-cm brown hair was conducted by GC-MS, LC-DAD and LC-MS/MS (0-2/2-4/4-6 cm; pg/mg). Haloperidol (9200/1391/227), amitriptyline (7450/1850/3260), venlafaxine (332/560/260), that had never been part of the victim's treatment were detected, as well as some benzodiazepines (alprazolam, bromazepam, nordazepam); cyamemazine was also detected in all the segments (9960/1610/2367) though only a single dose administration was reported in the medical records. The toxicological analyses performed at H0 and H48 confirmed the homicide attempts in the ICU. In addition, comparison of the results in hair analysis with the medical records confirmed repeated poisoning attempts over the previous six months, and thus explain the origin of the disorders presented by the victim. This case serves to remind us that repeated attempted murder can be difficult to diagnose and that hair analysis can be an effective way to detect such attempts.

Determination of compatibility and stability of haloperidol and morphine mixtures used in palliative care

Abstract With the aim of controlling various symptoms, possible to use mixtures of different drugs within infusion devices. This should take into account the compatibility of the mixture. Factors influence the compatibility and stability of the mixtures are: drug type, concentration, solvent, temperature and light. When evaluating the compatibility of the mixtures for infusion for subcutaneous via is important to consider infusion devices used and the conditions of light and temperature should simulate as far as possible the conditions in practice assistance. There are diverse studies that analyze the compatibility of drug mixtures, but there are still many possible combinations of drugs for which evidence is not available. The objective of this work is to study the compatibility and stability of several mixtures of haloperidol and morphine that can be used in solution for subcutaneous infusion.

Development of a new HPLC method for in vitro and in vivo studies of haloperidol in solid lipid nanoparticles

ABSTRACT A simple and sensitive HPLC method was developed and validated for the quantification of haloperidol in solid lipid nanoparticles (SLNs). The developed method was used for detection of shelf life of haloperidol in SLNs. Calibration curve of haloperidol was also constructed in rat plasma using loratidine as internal standard. In vivo studies were performed on rats and concentration of haloperidol in brain and blood was measured for the determination of various pharmacokinetic and hence brain targeting parameters. Chromatogram separation was achieved using C18 column as stationary phase. The mobile phase consisted of 100 mM/L potassium dihydrogen phosphate-acetonitrile-TEA (10:90:0.1, v/v/v) and the pH was adjusted with o-phosphoric acid to 3.5. Flow rate of mobile phase was 2 mL/minute and eluents were monitored at 230 nm using UV/VIS detector. The method was validated for linearity, precision, accuracy, reproducibility, limit of detection (LOD) and limit of quantification (LOQ). Linearity for haloperidol was in the range of 1-16 µg/mL. The value of LOD and LOQ was found to be 0.045 and 0.135 μg/mL respectively. The shelf life of SLNs formulation was found to be 2.31 years at 4 oC. Various parameters like drug targeting index (DTI), drug targeting efficiency (DTE) and nose-to-brain direct transport (DTP) were determined for HP-SLNs & HP-Sol administered intranasally to evaluate the extent of nose-to-brain delivery. The value of DTI, DTE and DTP for HP-SLNs was found to be 23.62, 2362.43 % and 95.77% while for HP-Sol, values were 11.28, 1128.61 % and 91.14 % respectively.

Facile stripping voltammetric determination of haloperidol using a high performance magnetite/carbon nanotube paste electrode in pharmaceutical and biological samples.

Multi-walled carbon nanotubes decorated with Fe3O4 nanoparticles were prepared to construct a novel sensor for the determination of haloperidol (Hp) by voltammetric methods. The morphology and properties of electrode surface were characterized by scanning electron microscopy (SEM) and electrochemical impedance spectroscopy. This modified sensor was used as a selective electrochemical sensor for the determination of trace amounts of Hp. The peak currents of differential pulse and square wave voltammograms of Hp increased linearly with its concentration in the ranges of 1.2×10(-3)-0.52 and 6.5×10(-4)-0.52µmol L(-1), respectively. The detection limits for Hp were 7.02×10(-4) and 1.33×10(-4)µmol L(-1) for differential pulse and square wave voltammetric methods, respectively. The results show that the combination of multi-walled carbon nanotubes and Fe3O4 nanoparticles causes a dramatic enhancement in the sensitivity of Hp quantification. This sensor was successfully applied to determine Hp in pharmaceutical samples and biological fluids. The fabricated electrode showed excellent reproducibility, repeatability and stability.

Monitoring haloperidol exposure in body fluids and hair of children by liquid chromatography-high-resolution mass spectrometry.

BACKGROUND: Haloperidol, 4-[4-(4-chlorophenyl)-4-hydroxy-1-piperidinyl]-1-(4-fluorophenyl)-1-butanone (HP), one of the most widely used antipsychotics in the treatment of schizophrenia, mania, and other psychiatric disorders, is frequently encountered in cases of unintentional pediatric intoxication because the ingestion of a small amount can cause significant toxic effects in children. For monitoring HP in suspected ingestions, a liquid chromatography-high-resolution mass spectrometry method has been developed and validated in urine, blood, and hair samples. METHODS: The analyte was extracted from 1 mL blood or urine by liquid/liquid extraction and from 5 mg of hair by micropulverized extraction; gradient elution on an Atlantis T3 column was realized using HP-d4 as an internal standard. Positive ion electrospray ionization and high-resolution mass spectrometry determination were performed in an Orbitrap mass spectrometer. RESULTS: The method exhibited a r > 0.999 in the studied ranges (0.1-50 ng/mL in urine and blood and 0.1-50 ng/mg in hair) and a limit of quantification of 0.1 ng/mL for urine and blood and 0.1 ng/mg for hair; intra-assay and interassay relative SDs were always more than 18%. The method was applied to determine haloperidol in 3 children who were admitted to emergency departments. HP concentrations ranged from 2 to 21 ng/mL in urine, from not detected to 4.9 ng/mL in blood, and from 0.37 to 0.73 ng/mg in hair samples. CONCLUSIONS: The utilization of high-resolution/high-accuracy mass spectrometry in full scan mode allowed the identification of HP metabolites in urine and blood, thus unequivocally documenting the exposure to the drug. HP metabolites were structurally characterized by high-resolution multiple mass spectrometry. For the first time, a HP metabolite was detected in hair.

Nano-electromembrane extraction.

The present work has for the first time described nano-electromembrane extraction (nano-EME). In nano-EME, five basic drugs substances were extracted as model analytes from 200 µL acidified sample solution, through a supported liquid membrane (SLM) of 2-nitrophenyl octyl ether (NPOE), and into approximately 8 nL phosphate buffer (pH 2.7) as acceptor phase. The driving force for the extraction was an electrical potential sustained over the SLM. The acceptor phase was located inside a fused silica capillary, and this capillary was also used for the final analysis of the acceptor phase by capillary electrophoresis (CE). In that way the sample preparation performed by nano-EME was coupled directly with a CE separation. Separation performance of 42,000-193,000 theoretical plates could easily be obtained by this direct sample preparation and injection technique that both provided enrichment as well as extraction selectivity. Compared with conventional EME, the acceptor phase volume in nano-EME was down-scaled by a factor of more than 1000. This resulted in a very high enrichment capacity. With loperamide as an example, an enrichment factor exceeding 500 was obtained in only 5 min of extraction. This corresponded to 100-times enrichment per minute of nano-EME. Nano-EME was found to be a very soft extraction technique, and about 99.2-99.9% of the analytes remained in the sample volume of 200 µL. The SLM could be reused for more than 200 nano-EME extractions, and memory effects in the membrane were avoided by effective electro-assisted cleaning, where the electrical potential was actively used to clean the membrane.

Evidence of Haldol (haloperidol) long-term intoxication.

A case of intoxication by haloperidol is reported. Haloperidol is a butyrophenone derivative commonly used in many hospital units as an antipsychotic agent. Adverse reactions due to haloperidol intoxication include drowsiness, blurred vision, extrapyramidal effects, tardive dyskinesia, tachycardia, hypotension and muscular rigidity. In August 2008, a 49 year-old female nurse started feeling various symptoms such as muscular rigidity, drowsiness and buccal dyskinesia. After 3 months, she was hospitalized for the worsening of these symptoms. Four months later, she showed once more the same symptoms. Two open water bottles from which the nurse used to drink in the hospital were confiscated and analyzed. Moreover, the nurse was asked to give a sample of her hair for executing the inherent toxicological analyses. Haloperidol was found in both bottles 1 and 2 at a concentration of 31.5 µg/mL and 43.6 µg/mL, respectively. Based on segmental hair analysis, it was deduced that the nurse consumed haloperidol in the approximate period from August 2008 to March 2009. The higher levels of haloperidol in hair were found in accordance with the periods of most severe appearance of symptoms, requiring the hospitalization of the nurse. The analysis of preservatives and excipients led us to conclude that the pharmaceutical drug was probably added to the water bottles as "Haldol 2mg/mL oral solution".

Stability studies of clonazepam, diazepam, haloperidol, and doxepin with diverse polarities in an acidic environment.

Stability of clonazepam, diazepam, haloperidol, and doxepin was determined in acidic solutions. In addition, determination of the kinetic and thermodynamic properties of this stability was carried out. Reaction rate constants (k), half-life times (t(0.1) and t(0.5)), and activation energy (Ea) were estimated for the drugs, which differed in polarity expressed with log P values. It was observed that estimated Ea values increased from 42.13 to 125.03 kJ/mol with an increase of lipophilicity (log P) beginning from the most hydrophilic drug (clonazepam, 2.70 log P) to the most lipophilic drug (doxepin, 4.10 log P). All degradation products were studied using an HPLC/electrospray ionization-MS technique in the positive ionization mode.

Development and validation of spectrofluorimetric method for determination of haloperidol in pharmaceutical dosage forms.

A simple, sensitive, and accurate spectrofluorimetric method has been developed for the determination of haloperidol in pharmaceutical preparations. The present method is based on the formation of an ion-pair complex between haloperidol and alizarin red S at pH 3.4 which is extractable with chloroform. The ion-pair complex exhibits maximum fluorescence intensity at 564 nm with excitation at 466 nm. The reaction conditions were optimized to obtain maximum fluorescence intensity. The relation between the fluorescence intensity and concentration was found to be linear over the range 0.8-20 microg/mL with detection limit of 0.08 microg/mL. The method was successfully employed for quantitation of the active ingredient haloperidol in pharmaceutical preparations. Statistical comparison of the results with the reference method shows excellent agreement and indicates no significant difference between the methods compared in terms of accuracy and precision.

Implementation of droplet-membrane-droplet liquid-phase microextraction under stagnant conditions for lab-on-a-chip applications.

In the current work, droplet-membrane-droplet liquid-phase microextraction (LPME) under totally stagnant conditions was presented for the first time. Subsequently, implementation of this concept on a microchip was demonstrated as a miniaturized, on-line sample preparation method. The performance level of the lab-on-a-chip system with integrated microextraction, capillary electrophoresis (CE) and laser-induced fluorescence (LIF) detection in a single miniaturized device was preliminarily investigated and characterized. Extractions under stagnant conditions were performed from 3.5 to 15 microL sample droplets, through a supported liquid membrane (SLM) sustained in the pores of a small piece of a flat polypropylene membrane, and into 3.5-15 microL of acceptor droplet. The basic model analytes pethidine, nortriptyline, methadone, haloperidol, and loperamide were extracted from alkaline sample droplets (pH 12), through 1-octanol as SLM, and into acidified acceptor droplets (pH 2) with recoveries ranging between 13 and 66% after 5 min of operation. For the acidic model analytes Bodipy FL C(5) and Oregon Green 488, the pH conditions were reversed, utilizing an acidic sample droplet and an alkaline acceptor droplet, and 1-octanol as SLM. As a result, recoveries for Bodipy FL C(5) and Oregon Green 488 from human urine were 15 and 25%, respectively.

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%.

Use of chemometrics for development and validation of an RP-HPLC method for simultaneous determination of haloperidol and related compounds.

A rapid resolution reversed-phase high performance liquid chromatographic (RR RP-HPLC) method has been developed and validated for simultaneous determination of haloperidol and six related compounds. Investigated matrix was a laboratory mixture of a therapeutic active substance haloperidol and its six related compounds in concentration ratio 300:1. Experimental design was used during method optimization (full factorial 23 design) and robustness testing (Central Composite Circumscribed design). Three factors: organic phase variation during gradient elution, flow rate and gradient rise time were independent variables. To estimate the system response during the optimization procedure and robustness testing, resolution (Rs) and a chromatographic response function (CRF) were used. Chromatography was performed with the mobile phase containing phosphate buffer pH 6.5 and acetonitrile as organic modifier. Separation was achieved using gradient elution (organic phase fraction changed linearly from 20 to 72 %) over 7 min. A Zorbax Eclipse XDB C18 Rapid Resolution HT 4.6 mm x 50 mm, 1.8 mum particle size, column was used at 25 degrees C at a flow rate of 1.5 mL min-1. UV detection was performed at 230 nm. The total time for chromatographic separation was 5.5 min with a total analysis time of 7.0 min. The method was validated for its linearity, precision, modal recovery and robustness.

Chemical stability of haloperidol injection by high performance thin-layer chromatography.

The chemical stability of haloperidol lactate injection was studied under different storage conditions by high performance thin-layer chromatography (HPTLC). The study was performed at 25 +/- 2 degrees C and at refrigeration temperature (8 +/- 1 degrees C) in original glass ampoules over 15 days after being opened. The samples tested at 25 +/- 2 degrees C were stored with exposure to and protection from light. Chromatographic separation was achieved on precoated silica gel F 254 HPTLC plates using a mixture of acetone/chloroform/n-butanol/glacial acetic acid/water (5:10:10:2.5:2.5, v/v/v/v/v) as a mobile phase. Quantitative analyses were carried out at a wavelength of 254 nm. The method exhibited adequate linearity (r = 0.999), selectivity, precision (RSD = 1.92%), and accuracy (recoveries from 98.59 to 101.90%). The concentrations of all samples remained greater than or 90% of the original concentration. Haloperidol lactate injection was chemically stable under all conditions studied over 15 days.

Sensitive liquid chromatography/tandem mass spectrometry method for the simultaneous determination of olanzapine, risperidone, 9-hydroxyrisperidone, clozapine, haloperidol and ziprasidone in rat brain tissue.

One prerequisite for therapeutic effects of psychiatric drugs is the ability to pass the blood brain barrier. Hence, it is important to know the concentration of antipsychotic drugs in brain tissue. In general, determinations of lipophilic compounds from lipophilic matricies such as the brain are a challenge. Here we have adapted a plasma assay for antipsychotics for the target organ the brain. Using modified sample preparation and chromatographic strategies, the analytes were extracted from rat brain homogenate and analyzed by LC-MS/MS. The method used a Waters Atlantis dC-18 (30 mm x 2.1 mm i.d., 3 microm) column with a mobile phase of acetonitrile/5 mM ammonium formate (pH 6.1 adjusted with formic acid) and gradient elution. All analytes were detected in positive ion mode using multiple-reaction monitoring. The method was validated and the linearity, lower limit of quantitation, precision, accuracy, recoveries, specificity and stability were determined. This method was then successfully used to quantify the rat brain tissue concentration of the analytes after chronic treatment with these antipsychotic drugs.

Properties of C84 and C24H12 molecular ion sources for routine TOF-SIMS analysis.

C84+ and coronene (C24H12+) have been studied as primary ions for use in secondary ion mass spectrometry. A representative range of samples has been used to compare the effectiveness of each primary ion with the existing C60+, Au+, and Au3+ primary ions. It was found that C84 is the most effective primary ion providing higher secondary ion yields and a high molecular to fragment ion ratio. Coronene had a performance similar to C60. Coronene and C60 primary ions were also used to extend a previous study of matrix suppression/enhancement effects. The C60 was found to ameliorate this effect, possibly due to the increase in protonation in polyatomic sputtering, and coronene was found to further reduce suppression showing evidence of a chemical effect.

An on-chip cardiomyocyte cell network assay for stable drug screening regarding community effect of cell network size.

We investigate the effect of haloperidol on a four-cell and nine-cell cardiomyocyte network on an agarose microchamber array chip to evaluate a cell-based model for drug screening. Using a network of cardiomyocytes whose beating intervals were stable and relatively uniform (they only fluctuated 10% from the mean beating interval), we easily observed the effect of haloperidol on the cell network beating interval 5 min after administering it. We also observed the beating interval returned to its original state 10 min after the haloperidol was washed out of the chip. Although the four-cell network showed the unstable recovery of its beating rhythm after washout of haloperidol, the nine-cell network recovered completely to the stable original beating rhythm even after a second administration of haloperidol. The results indicate the importance of the community size in cell networks used in the stable cell-based screening model. Moreover, they indicate the advantage of using direct cell-based measurements in which the amount of drug administered and the time course over which it is administered are strictly controlled for evaluating the quantitative chemical effects of drugs on cells.

Quantitative determination of haloperidol in tablets by high performance thin-layer chromatography.

A densitometric high performance thin-layer chromatography (HPTLC) method was developed and validated for the quantitative analysis of haloperidol in tablets. Chromatographic separation was achieved on precoated silica gel F 254 HPTLC plates using a mixture of acetone/chloroform/n-butanol/acetic acid glacial/water (5:10:10:2.5:2.5 v/v/v/v/v) as the mobile phase. Quantitative analysis was carried out at a wavelength of 254 nm. The method was linear in the 10-100 ng/microL range, with a determination coefficient of 0.999. The coefficients of variation for precision were not higher than 2.35%. The detection limit was 0.89 ng/microL, and the quantification limit was 2.71 ng/microL. The accuracy ranged from 97.76 to 100.33%, with a CV not higher than 4.50%. This method was successfully applied to quantify haloperidol in real pharmaceutical samples, including the comparison with HPLC measurements. The method was fast, specific, with a good precision and accuracy for the quantitative determination of haloperidol in tablets.