Facile synthesis of magnetic molecularly imprinted polymer: Perphenazine template and its application in urine and plasma analysis.

Synthesis of magnetic iron oxide nanoparticles and its surface modification with methacrylic acid (MAA) was performed simultaneously by adding Fe(2+)/Fe(3+) to an alkaline MAA solution under nitrogen atmosphere. MAA coated magnetite (Fe3O4@MAA) has abundant reactive double bonds on the surface that can initiate polymerization. Magnetic molecularly imprinted polymers (MMIPs) were synthesized through distillation-precipitation polymerization of MAA as monomer, perphenazine (PPZ) as template, and ethylene glycol di-methacrylate (EGDMA) as cross linker on Fe3O4@MAA, with concise control of experimental conditions in about 90min. The produced super paramagnetic MMIPs can be separated from the solution in the presence of external magnetic field in less than 1min. Characterizations of the synthesized particles were performed by electron microscopes, thermo-gravimetric analysis (TGA), vibrating sample magnetometer (VSM), Fourier transform infrared (FT-IR) spectroscopy, and BET. The data showed that Fe3O4@MAA was well encapsulated in the polymer shell. The MMIPs showed high porosity. Moreover, MMIPs were used for rapid pre-concentration and separation of PPZ in human plasma and urine without any dilution and pretreatments using high performance liquid chromatography equipped with a photo diode array detector (HPLC-PDA). The calibration curve in urine and plasma has shown the same slope as the external calibration curve. Linear range of 20-5000ngmL(-1), and a detection limit of 5.3ngmL(-1) was obtained. The results showed 97.92% recovery along with the relative standard deviation of 6.07% (n=6) for 1µgmL(-1) PPZ. Pre-concentration factor was 13. The MMIPs adsorbed PPZ in 1min and then desorbed it by MeOH:HOAc in 2min.

Quantitation of Haloperidol, Fluphenazine, Perphenazine, and Thiothixene in Serum or Plasma Using Liquid Chromatography-Tandem Mass Spectrometry (LC-MS/MS).

Haloperidol, fluphenazine, perphenazine, and thiothixene are "typical" antipsychotic drugs that are used in the treatment of schizophrenia and other psychiatric disorders. The monitoring of the use of these drugs has applications in therapeutic drug monitoring and overdose situations. LC-MS/MS is used to analyze plasma/serum extracts with deuterated analog of imipramine as the internal standard to ensure accurate quantitation and control for any potential matrix effects. Positive ion electrospray is used to introduce the analytes into the mass spectrometer. Selected reaction monitoring of two product ions for each analyte allows for the calculation of ion ratios which ensures correct identification of each analyte, while a matrix-matched calibration curve is used for quantitation.

Simultaneous UPLC-MS/MS assay for the detection of the traditional antipsychotics haloperidol, fluphenazine, perphenazine, and thiothixene in serum and plasma.

BACKGROUND: Most antipsychotic drugs that are commonly prescribed in the USA are monitored by liquid and gas chromatographic methods. Method performance has been improved using ultra high pressure liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS). A rapid and simple procedure for monitoring haloperidol, thiothixene, fluphenazine, and perphenazine is described here. METHOD: Antipsychotic drug concentrations in serum and plasma were determined by LCMS/MS (Waters Acquity UPLC TQD). The instrument is operated with an ESI interface, in multiple reaction monitoring (MRM), and positive ion mode. The resolution of both quadrupoles was maintained at unit mass with a peak width at half height of 0.7amu. Data analysis was performed using the Waters Quanlynx software. Serum or plasma samples were thawed at room temperature and a 100µL aliquot was placed in a tube. Then 300µL of precipitating reagent (acetonitrile-methanol [50:50, volume: volume]) containing the internal standard (0.12ng/µL Imipramine-D3) was added to each tube. The samples were vortexed and centrifuged. The supernatant was transferred to an autosampler vial and 8µL was injected into the UPLC-MS/MS. Utilizing a Waters Acquity UPLC HSS T3 1.8µm, 2.1×50mm column at 25ºC, the analytes were separated using a timed, linear gradient of acetonitrile and water, each having 0.1% formic acid added. The column is eluted into the LC-MS/MS to detect imipramine D3 at transition 284.25>89.10, haloperidol at 376.18>165.06, thiothixene at 444.27>139.24, fluphenazine at 438.27>171.11, and perphenazine at 404.19>143.07. Secondary transitions for each analyte are also monitored for imipramine D3 at 284.25>193.10, haloperidol at 376.18>122.97, thiothixene at 444.27>97.93, fluphenazine at 438.27>143.08, and perphenazine at 404.19>171.11. The run-time is 1.8min per injection with baseline resolved chromatographic separation. RESULTS: The analytical measurement range was 0.2 to 12.0ng/mL for fluphenazine and perphenazine, and was 1 to 60.0ng/mL for haloperidol and thiothixene. Intra-assay and inter-assay imprecisions (CV) were less than 15% at two concentrations for each analyte. CONCLUSIONS: By utilizing a LC-MS/MS method we combined two previously established analytical assays into one, yielding a 75% time-savings on set-up, and a significantly shortened analytical run-time. These changes reduced the turn-around time for analysis and eliminated interference issues resulting in fewer injections and increased column lifetime.

BL-1020, a novel antipsychotic candidate with GABA-enhancing effects: D2 receptor occupancy study in humans.

BL-1020 is a potentially novel antipsychotic, which comprises the typical antipsychotic perphenazine linked by an ester bound to gamma-aminobutyric acid (GABA), intending a simultaneous dopamine-2 (D(2)) receptor blockade and GABA facilitation in the brain. This positron emission tomography (PET) study, using [(11)C]raclopride, assessed the extent and duration of D(2) receptor occupancy (D(2) RO) and safety for single doses of BL-1020 in healthy male subjects. Overall, this study did not raise any safety concern. Single doses of 16-32 mg BL-1020 caused a dose dependent striatal D(2) RO. The 32 mg dose of BL-1020 resulted in an average D(2) RO of 44% at 4-6 h post dosing (pd), which declined to 33% at 24 h pd. Equimolar doses of BL-1020 and perphenazine resulted in similar D(2) RO at 24 h pd. Pharmacokinetic-pharmacodynamic analysis predicted that oral once daily administration of 32 mg BL-1020 would result in D(2) ROs ranging from 52 to 66% at a steady state.

Spectrophotometric determination of perphenazine with the detection system of potassium ferricyanide-Fe(III) in pharmaceutical and serum samples.

In this paper, a novel spectrophotometric method has been established to determine perphenazine using the detection system of potassium ferricyanide-Fe(III). In the presence of potassium ferricyanide, it has been demonstrated that Fe(III) is reduced to Fe(II) by perphenazine at pH 4.0. In addition, soluble prussian blue (KFe(III)[Fe(II)(CN)(6)]) is produced by the reaction between the in situ formed Fe(II) and potassium ferricyanide. The absorbance of soluble prussian blue is measured at the absorption maximum of 735 nm, and the amount of perphenazine can be calculated based on this absorbance. The absorbance is linear to perphenazine concentration in the ranges of 0.05-25.00 microg/mL with correlation coefficients of 0.9997, and the linear regression equation is A = 0.0069 + 0.1285C (microg/mL). The detection limit (3sigma/k) is 0.049 microg/mL, and the relative standard deviation (R.S.D.) is 0.86% (n = 11). Moreover, the apparent molar absorption coefficient of indirect determination of perphenazine is 5.2 x 10(4) L/mol cm. The parameters with regard to determination are optimized, and the reaction mechanism is discussed. This method has been successfully applied to the determination of perphenazine in pharmaceutical and serum samples. Analytical results obtained with this novel assay are satisfactory.

Development and validation of a gas chromatographic-mass spectrometric method for quantitative determination of perphenazine in rabbit plasma after sublingual administration.

Perphenazine is a phenothiazine-type antipsychotic that is a potential candidate for sublingual administration due to its extensive first-pass metabolism. In this study, a gas chromatographic-mass spectrometric method was developed for quantification of perphenazine in rabbit plasma after sublingual administration. The plasma samples were purified by mixed-mode solid phase extraction with good recovery (>83%). The method was linear (r(2)>0.99) over a range of 2-64 ng/ml, with a lower limit of quantification of 2 ng/ml. The accuracy was 100+/-4%, and the within-day and between-day precisions were <6.8% R.S.D. and <14% R.S.D., respectively. Perphenazine was stable in stock solutions and plasma. The method was successfully applied for analysing perphenazine in plasma after sublingual administration to rabbits.

HPLC quantification of perphenazine in sheep plasma: application to a pharmacokinetic study.

A high performance liquid chromatographic (HPLC) method for the determination of perphenazine (PPZ) in sheep plasma was developed and validated. The separation was achieved using a 5 microm C18 column (125 mm x 4 mm) with a mobile phase composed of acetonitrile and an aqueous solution of H(3)PO(4) and TBA (inverse gradient). The flow rate was 1.5 mL/min and the UV detection was performed at 258 nm. The method was validated with respect to linearity, intra and inter-day precision and accuracy, limit of quantification, limit of detection and storage stability. This method was used to perform a pilot pharmacokinetic study of PPZ after subcutaneous administration to one ewe.

A cross-validation study on the relationship between central D2 receptor occupancy and serum perphenazine concentration.

RATIONALE: There is a need for laboratory measures to guide clinical treatment with antipsychotic drugs. For serum concentration of the classical antipsychotic drug perphenzine an optimal therapeutic interval has been identified between 2 and 6 nmol/l. Positron emission tomography (PET) studies have suggested an optimal interval in central dopamine D2 receptor occupancy of between 65 and 80%. OBJECTIVES: The aim of the present cross-validation study in clinically stable schizophrenic patients was to examine the relationship between the optimal interval in central D2 receptor occupancy and the therapeutic window for serum perphenazine concentration. METHODS: Six patients who had responded to maintenance treatment with perphenazine decanoate were examined with PET and [11C]raclopride during steady-state conditions. Blood sampling was carried out for minimum serum perphenazine concentration and during the PET examination. RESULTS. The serum perphenazine concentration was between 1.8 and 9 nmol/l and the D2 receptor occupancy varied between 66 and 82%. The relationship between central receptor occupancy and serum drug concentration was curvilinear. Mild extrapyramidal symptoms were present in the patient with the highest D2 receptor occupancy. CONCLUSIONS. The previously suggested therapeutic window in serum perphenazine concentration is in good agreement with the optimal interval suggested for central D2 receptor occupancy. Serum concentrations at low dose levels may therefore serve as a useful tool in clinical monitoring of antipsychotic drug treatment.

Antipsychotic radioreceptor assay: a modification identifying selective receptor effects.

Radioreceptor assays offer the advantage of a single assay that can assess uniform exposure to multiple chemical compounds. The advent of atypical antipsychotic agents has led to new awareness of the multiple receptor subtypes through which antipsychotic agents may exert their effects, and a renewed interest in comparative drug trials of antipsychotics. The objective of this study was to show the development and validation of antipsychotic radioreceptor assays using clonal cell lines stably expressing isolated human receptors. Model assays were developed using the dopamine(2) (D(2)) and D(4) receptors. D(2) and D(4) activities measured by radioreceptor assay in plasma of antipsychotic-treated subjects were highly correlated with high-performance liquid chromatography determinations of antipsychotic concentrations. Similarly, for a variety of typical and atypical antipsychotic agents, the quotients of D(4)/D(2) activity in plasma of antipsychotic-treated subjects were highly correlated with the quotients of D(4)/D(2) affinities of these agents. Valid receptor-selective antipsychotic assays can be established and may have utility for dissecting the in vivo activity of atypical antipsychotics in relation to specific outcomes in clinical trials.

Therapeutic drug monitoring of haloperidol, perphenazine, and zuclopenthixol in serum by a fully automated sequential solid phase extraction followed by high-performance liquid chromatography.

In Denmark, haloperidol, perphenazine, and zuclopenthixol are among the most frequently requested antipsychotics for therapeutic drug monitoring. With the number of requests made at the authors' laboratory, the only rational analysis is one that can measure all three drugs simultaneously. The authors therefore decided to develop an automated high-performance liquid chromatography (HPLC) method. Two milliliters serum, 2.0 mL 10 mmol/L sodium phosphate buffer (pH 5.5), and 150 microL internal standard (trifluoperazine) solution were pipetted into HPLC vials and extracted on an ASPEC XL equipped with 1 mL (50 mg) Isolute C2 (EC) extraction columns and acetonitrile-methanol-ammonium acetate buffer (60:34:6) as extracting solution. Three hundred fifty microliters was analyzed by HPLC; a 150 x 4.6-mm S5CN Spherisorb column with a mobile phase of 10 mmol/L ammonium acetate buffer-methanol (1:9), a flow rate of 0.6-1.7 mL/min, and ultraviolet detection at 256 and 245 nm were used. Reproducibility was 5-12% and the lower limit of quantitation was 10, 1, and 5 nmol/L (4, 0.4, and 2 ng/mL) for haloperidol, perphenazine, and zuclopenthixol, respectively. The method was found to be sufficiently selective and robust for routine analysis.

Perphenazine distribution in a postmortem case.

The case of a 34-year-old, mentally challenged, Caucasian female found dead in a group home is presented. Empty containers of perphenazine and valproic acid were found next to her bed. The perphenazine had been prescribed to another patient. No anatomic cause of death was determined at autopsy. Comprehensive testing of the heart blood for ethanol and drugs identified perphenazine at a concentration of 4.4 mg/L and valproic acid at a concentration of 950 mg/L. The distribution of perphenazine in other specimens was consistent with previously reported phenothiazine cases. The medical examiner ruled that the cause of death in this case was multiple drug intoxication and the manner of death was suicide.

Capillary zone electrophoresis determination of perphenazine in pharmaceutical preparations and in human serum.

Perphenazine belongs to the group of phenothiazine-based neuroleptic drugs frequently used in the long-term treatment of psychotic disorders. In this work, a new capillary zone electrophoresis (CZE) method for the rapid determination of perphenazine (PPZ) in pharmaceutical preparations and human blood serum was developed. Using solid-phase extraction (SPE) as a preclean/purification and preconcentration step before CZE analysis, a detection limit of 3 ng/mL for the monitoring of PPZ in blood serum (for a signal-to-noise ratio of 3) was reached.

Acute neuroleptic treatment in elderly patients without dementia.

Low doses of neuroleptics are the standard for treating psychosis in elderly patients because of concern about inducing adverse effects. The authors found that fixed, low-dose neuroleptic treatment (0.15 mg/kg/day) for 10 days resulted in low perphenazine levels and low rates of acute response (25%) in elderly patients with primary psychotic illness (without dementia). Increase in initial dose did not speed acute response and induced adverse effects that were absent or minimal with low-dose treatment. With higher-dose treatment, drug blood levels rose disproportionately, and level-to-dose ratios were higher than those observed in non-elderly adults. Naturalistic follow-up suggested that response may take longer to develop than in non-elderly adults and that low doses for a longer duration may provide effective treatment.

[Determination of basic drugs in blood by RP-HPLC].

A reversed-phase HPLC method for determination of phenothiazines and tricyclin antidepressants in whole blood was described. In this paper, a model 1090 HPLC with DAD and a zorbax ODS column was used. The mobile phase was methanol: water: tritely amine (75:24.7:0.3) with pH 7.5. Cyproheptadine was used as internal standard in this method. Blood samples were extracted with the solid-phase extraction method and the liquid-liquid method. This method is suitable in forensic toxicology analysis for basic drugs.

Steady-state serum concentrations of the neuroleptic perphenazine in relation to CYP2D6 genetic polymorphism.

Steady-state serum concentration to dose ratios of the neuroleptic agent perphenazine were related to CYP2D6 metabolizer status for 96 psychiatric inpatients: 88 extensive metabolizers and eight poor metabolizers. The median concentration per dose of the poor metabolizer group (0.195 nmol/L per milligram) was about twice the median (0.098 nmol/L per milligram) of the 56 extensive metabolizers without interacting medicine (p < 0.01). The rest of the extensive metabolizers (n = 32), who were comedicated with drugs that compete with perphenazine for metabolism by CYP2D6, had an intermediate median value of 0.140 nmol/L per milligram. The range of concentration/dose values for the total extensive metabolizer group extended from 0.025 to 0.688 nmol/L per milligram, that is, an almost thirtyfold variation. The concentration/dose range of the eight poor metabolizer subjects was 0.096 to 0.750 nmol/L per milligram. Serum levels not corrected for dose overlapped to a large degree among the groups, with a total range from 0.5 to 12 nmol/L. This study points toward a limited information value of CYP2D6 genotyping in the context of therapeutic drug monitoring of perphenazine.

Quantitative determination of perphenazine and its metabolites in plasma by high-performance liquid chromatography and coulometric detection.

An accurate, reliable method has been developed for the therapeutic monitoring of perphenazine (PPZ) and its major metabolites in human plasma samples. Steady-state plasma levels of PPZ and its metabolites were quantitated for 30 elderly patients (mean age: 75) undergoing concurrent treatment with nortriptyline (NT) and PPZ, doses ranging from 4 to 32 mg/day for PPZ. The assay was suitable with patients on concurrent medications, and smaller patient plasma volumes (1 ml) were used indicating sufficient sensitivity and specificity. After plasma extraction and separation on a Nucleosil 5-microns C18 column, the recoveries (mean +/- S.D.) of PPZ and its metabolites were determined; perphenazine 92 +/- 7.5%, deshydroxyethylperphenazine 81 +/- 7.2%, perphenazine sulfoxide 68 +/- 6.4%, and 7-hydroxyperphenazine 45 +/- 5.5%. The assay also had limits of quantitative detectability for PPZ and its metabolites as follows: perphenazine 0.5 ng/ml, deshydroxyethylperphenazine 1.0 ng/ml, perphenazine sulfoxide 0.5 ng/ml, and 7-hydroxyperphenazine 5 ng/ml. Inter-assay reproducibility (C.V.) for the quality controls and patient samples ranged from 18.8 to 2.4%. The sensitivity and reproducibility of this method should improve PPZ therapeutic drug monitoring and research on interactions in depressed geriatric patients.