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Cell-based screening systems for pharmaceuticals are desired over molecular biosensing systems because of the information they provide on toxicity and bioavailability. However, the majority of sensing systems developed are molecular biosensing type screening systems and cannot be easily adapted to cell-based screening. In this study, we demonstrate that protein-based molecular sensing systems that employ a fluorescent protein as a signal transducer are amenable to cell-based sensing by expressing the protein molecular sensing system in the cell and employing these cells for screening of desired molecules. To achieve this, we expressed a molecular sensing system based on the fusion protein of calmodulin (CaM) and enhanced green fluorescent protein (EGFP) in bacterial cells, and utilized these cells for the screening of CaM antagonists. In the presence of Ca(2+), CaM undergoes a conformational change exposing a hydrophobic pocket that interacts with CaM-binding proteins, peptides, and drugs. This conformational change induced in CaM leads to a change in the microenvironment of EGFP, resulting in a change in its fluorescence intensity. The observed change in fluorescence intensity of EGFP can be correlated to the concentration of the analyte present in the sample. Dose-response curves for various tricyclic antidepressants were generated using cells containing CaM-EGFP fusion protein. Additionally, we demonstrate the versatility of our system for studying protein-protein interactions by using cells to study the binding of a peptide to CaM. The study showed that the CaM-EGFP fusion protein within the intact cells responds similarly to that of the isolated fusion protein, hence eliminating the need for any isolation and purification steps. We have demonstrated that this system can be used for the rapid screening of various CaM antagonists that are potential antipsychotic drugs.

HPLC is discussed as an essential tool in medicinal chemistry for the monitoring of tricyclic antidepressants in biofluids, providing clinicians with efficient fast and reliable methods to define individual optimum therapeutic concentrations in treatment of depressions. Additional information on mechanism of action, structure activity relationship and metabolism is provided.

A simple and rapid spectrophotometric method has been developed for the determination of tricyclic anti-depressant drugs such as trazodone (TZH), amineptine (APH) and amitriptyline (ATPH) hydrochlorides in pure form and in different pharmaceutical preparations. The charge transfer (CT) reaction between TZH, APH and ATPH as electron donors and TCNQ as electron acceptor was utilized for their spectrophotometric determination. The optimum experimental conditions, like time, temperature, stoichiometry, solvents, for the CT complex formation are established. The method permits the determination of TZH, APH and ATPH over a concentration range of 10-400, 10-440 and 10-300 microg ml(-1), respectively. The sensitivity (S) is found to be 0.09, 0.087 and 0.069 g cm(-2) for TZH, APH and ATPH, respectively. The SD values are found to be 0.146-0.293, 0.154-0.285 and 0.091-0.212 and RSD values are 0.142-1.92, 0.297-1.92 and 0.212-0.915 for TZH, APH and ATPH, respectively. The low values of the relative standard deviation indicate the high accuracy and precision of the method. The mean recovery values obtained together with a high correlation coefficient values, amount in the range 98-101.5, 98.7-102.9 and 93-101.9 for TZH, APH and ATPH, respectively. The method is applicable for the assay of the investigated drugs in different dosage forms and the results are in good agreement with those obtained by the official method.

Two simple, sensitive and rapid extractive spectrophotometric methods have been developed for the assay of the antidepressant drug nortriptyline (NOR) hydrochloride in pure form and in different dosage forms. The methods involve the formation of colored ion-pairs between the drug and the complex of niobium(V)-thiocyanate (Nb-SCN) or iron(III)-thiocyanate (Fe-SCN) followed by their extraction with butanol or a mixture of butanol and chloroform and quantitative determination at 360 nm and 490 nm, using Nb-SCN and Fe-SCN, respectively. The experimental conditions were optimized to obtain the maximum colour intensity. The methods permit the determination of nortriptyline over a concentration range of 15-100 microg/ml and 5-24 microg/ml with the detection limit of 0.84 microg/ml and 0.32 microg/ml, using Nb-SCN and Fe-SCN, respectively. The proposed methods are applicable for the assay of the investigated drug in different dosage forms and the results are in good agreement with those obtained by the official and HPLC methods. No interference was observed from common excipients present in pharmaceutical formulations. The proposed procedures were applied to determine the amount of nortriptyline hydrochloride as active ingredient in the presence of its degradation product, dibenzosuberone. The extractive spectrophotometric methods can also be used to determine the amount of nortriptyline hydrochloride in tablets after its solid phase extraction (SPE).

A simple, rapid and sensitive HPLC method was developed and validated for the determination of four tricyclic antidepressants (TCAs): amitriptyline, doxepin, clomipramine (CLO) and imipramine, in pharmaceutical formulations and biological fluids. A Kromasil C(8 )analytical column (250 x 4 mm, 5 microm) was used for the separation, with a mobile phase consisting of 0.05 M CH(3)COONH(4) and CH(3)CN (45:55 v/v) delivered at 1.5 mL/min isocratically. Quantification was performed at 238 nm, with bromazepam (1.5 ng/microL) as the internal standard. The determination of TCAs in blood plasma was performed after protein precipitation. Urine analysis was performed by means of SPE using Lichrolut RP-18 Merck cartridges providing high absolute recoveries (> 94%). Direct analysis of urine was also performed after two-fold dilution. The developed method was fully validated in terms of selectivity, linearity, accuracy, precision, stability and sensitivity. Repeatability (n = 5) and between-day precision (n = 5) revealed RSD <13%. Recoveries from biological samples ranged from 91.0 to 114.0%. The absolute detection limit of the method was calculated as 0.1-0.6 ng in blood plasma and 0.2-0.5 ng in extracted urine or 0.4-0.7 in diluted urine. The method was applied to real samples of plasma from a patient under CLO treatment.

Cyclodextrins (CDs) are widely used in the pharmaceutical industry for their capability of improving bioavailability, solubility, or stability of drugs via the formation of soluble inclusion complexes. CDs have also been widely used in various chemical analysis methods. In this work, liquid chromatography/electrospray mass spectrometry (LC/ESI-MS) analysis for four different drugs (imipramine, desipramine, propranolol, and naproxen) that form inclusion complexes with CDs was performed in the presence and absence of beta-CD. These drugs are subject to nonspecific adsorption when brought into contact with plastics, such as HPLC tubing, sample collection and preparation apparatus, etc. Inclusion of the CD in the samples reduces this nonspecific adsorption due to competitive complex formation between the CD and the analyte. ESI-MS ion intensities increased when beta-CD was included in the sample with concentrations up to 1% (w:v), with a diverter valve installed post LC column. The degree of increased ion signal correlated with the beta-cyclodextrin:analyte binding constant. beta-CD appeared to elute within the void volume time and was observed in a full spectrum scan among the different analyte samples with up to 0.01% beta-CD injected directly to the LC/MS system with the diverter valve switched inline with the mass spectrometer. The use of the diverter valve allowed for direct injection of samples containing up to 1% beta-CD to the LC/MS without any deterioration of analyte ion signal.

A new high-performance liquid chromatography method is presented for the determination of 10 frequently prescribed tricyclic and nontricyclic antidepressants: imipramine, amitriptyline, clomipramine, fluoxetine, sertraline, paroxetine, citalopram, mirtazapine, moclobemide and duloxetine. The simple and accurate sample preparation step, consisted of liquid:liquid extraction with recoveries ranging between 72% and 86%, except for moclobemide (59%). Separation was obtained using a reverse phase Select B column under isocratic conditions with UV detection (230 nm). The mobile phase consisted of 35% of a mixture of acetonitrile/methanol (92:8, v/v) and 65% of 0.25 mol L(-1) sodium acetate buffer, pH 4.5. The standard curves were linear over a working range of 2.5-1000 ng mL(-1) for moclobemide, 5-2000 ng mL(-1) for citalopram, duloxetine, fluoxetine, 10-2000 ng mL(-1) for sertraline, imipramine, paroxetine, mirtazapine and clomipramine. The intra-assay and inter-assay precision and accuracy were studied at three concentrations (50, 200, and 500 ng mL(-1)). The intra-assay coefficients of variation (CVs) for all compounds were less than 8.8%, and all inter-CVs were less than 10%. Limits of quantification were 2.5 ng mL(-1) for moclobemide, 5 ng mL(-1) for citalopram, duloxetine and amitriptyline, and 10 ng mL(-1) for mirtazapine, paroxetine, imipramine, fluoxetine, sertraline, and clomipramine. No interference of the drugs normally associated with antidepressants was observed. The method has been successfully applied to the analysis of real samples, for the drug monitoring of ten frequently prescribed tricyclic and non-tricyclic antidepressant drugs.

Tianeptine (Stablon), although structurally similar to tricyclic antidepressants, acts by enhancing the reuptake of serotonin. A fatal case is presented involving a 26-year-old man, found lying in bed with a "mushroom of foam" around his mouth. Empty blister packs of Stablon and a suicide note were found next to the body. A liquid-liquid extraction procedure with n-hexane: ethyl acetate and n-hexane: 2-propanol, followed by LC-DAD-MS analysis, using positive mode electrospray ionization was performed. The detection limit was 0.001 microg/mL. The toxicological results revealed the following tianeptine concentrations in the post-mortem samples: blood 5.1 microg/mL; urine 2.0 microg/mL; liver 23 microg/g; stomach contents 22 mg. Femoral blood analyses also revealed an ethanol concentration of 0.53 g/L. The present method was also developed and validated for the other post-mortem specimens, since no previous published data had confirmed the post-mortem distribution of tianeptine. The absence of other suitable direct causes of death (macroscopic or histological) and the positive results achieved with the toxicological analysis led the pathologist to rule that death was due to an intoxication caused by the suicidal ingestion of tianeptine in combination with alcohol.

The purpose of this work is to develop a sensitive, selective, and validated stability-indicating HPLC assay of tianeptine (TIA) in bulk drug and tablet form. TIA is subjected to different stress conditions, including UV-light, oxidation, acid base-base hydrolysis, and temperature. TIA and its possible degradation products are analyzed on Agilent-Zorbax-XDB-C18 column using gradient elution with acetonitrile and 0.02M sodium acetate (pH 4.2). The samples are monitored simultaneously with photo-diode array at 254 nm and fluoroscence detector set to 350 nm (ex) and 425 nm (em). TIA is integrated from its UV-chromatogram, and the photodecomposition products are integrated from the fluoroscence-chromatogram. TIA and its photodecomposition products are separated by TLC using ethyl acetate-n-hexane-glacial acetic acid-methanol (10.0:14.0:0.2:1.0, v/v) as developing system. One potential photodegradation product is detected by fluoroscence in TIA-tablet form and separated by TLC. The linear range of TIA is between 0.5 to 50 microg/injection with limits of quantitation and detection values of 30 and 8 ng/injection, respectively. The inter-assay percentage of deviation is not more than 0.03%, and the day-to-day variation is not more than 0.1%.

A sensitive and selective high-performance liquid chromatographic method has been developed for the determination of tianeptine (Tia) in tablets. The method is based on derivatization of Tia with 4-chloro-7-nitrobenzofurazan (NBD-CI). A mobile phase consisting of acetonitrile-10 mM orthophosphoric acid (pH 2.5; 77 + 23) was used at a flow rate of 1 mL/min on a C18 column. The Tia-NBD derivative was monitored using a fluorescence detector, with emission set at 520 nm and excitation at 458 nm. Gabapentin was selected as an internal standard. Linear calibration graphs were obtained in the concentration range of 45-300 ng/mL. The lower limit of detection (LOD) was 10 ng/mL at a signal-to-noise ratio of 4. The lower limit of quantitation (LOQ) was 45 ng/mL. The relative standard values for intra- and interday precision were <0.46 and <0.57%, respectively. The recovery of the drug samples ranged between 98.89 and 99.85%. No chromatographic interference from the tablet excipients was found. The proposed method was validated in terms of precision, robustness, recovery, LOD, and LOQ. All the validation parameters were within the acceptance range. The proposed method was applied for the determination of Tia in commercially available tablets. The results were compared with those obtained by an ultraviolet spectrophotometric method using t- and F-tests.

A reversed-phase liquid chromatographic method has been developed for the detection and control of impurities of imipramine hydrochloride at a level of 0.2 microg/ml (corresponding to 0.02 per cent in the procedure described). This method is included in a proposed revision of the monograph (Pharmeuropa 18.4) and replaces the thin-layer chromatographic method currently employed.

The work presented here explores the possibilities of the electrokinetic injection (EK) to achieve sensitive methods for the determination of tricyclic antidepressants in biological samples (serum). The addition of ACN to the sample, with high content in salts, causes stacking at the tip of the capillary, in a similar way as for hydrodynamic injection. An experimental design with the response surface methodology has been used to find the optimum composition of the matrix of the sample (sodium chloride and ACN percentages) and the conditions for the EK (water-plug length, time, and voltage of injection) in few experiments. The composition of the separation buffer was the same as utilized in a previous paper. The use of a bubble capillary to reach lower detection limits implies a loss of the resolution and requires a new optimization. Finally, a comparison between electrokinetic and hydrodynamic injections is made.

Differential pulse polarographic (DPP) and square wave polarographic (SWP) techniques were applied at hanging mercury drop electrode (HMDE) for quantitative determination of tianeptine (TIA) in tablets. The adsorptive stripping voltammetric (ASV) behavior of TIA was also studied. TIA gave a sensitive reduction peaks at -1256, -1244 and -1072 mV for DPP, SWP and ASV, respectively (versus Ag/AgCl) in Britton-Robinson buffer (B-R buffer) at pH 11. The solution conditions and instrumental parameters were optimized for the determination of TIA in tablets. Calibration plots and regression data validation, accuracy, precision, limit of detection, limit of quantitation and other aspects of analytical merit are presented.

Literature data relevant to the decision to allow a waiver of in vivo bioequivalence (BE) testing for the approval of immediate release (IR) solid oral dosage forms containing amitriptyline hydrochloride are reviewed. Its therapeutic uses, its pharmacokinetic properties, the possibility of excipient interactions and reported BE/bioavailability (BA) problems are also taken into consideration. Literature data indicates that amitriptyline hydrochloride is a highly permeable active pharmaceutical ingredient (API). Data on the solubility according to the current Biopharmaceutics Classification System (BCS) were not fully available and consequently amitriptyline hydrochloride could not be definitively assigned to either BCS Class I or BCS Class II. But all evidence taken together, a biowaiver can currently be recommended provided that IR tablets are formulated with excipients used in existing approved products and that the dissolution meets the criteria defined in the Guidances., ((c) 2006 Wiley-Liss, Inc. and the American Pharmacists Association)

Objectives: Patients in emergency departments who use methadone frequently use tricyclic antidepressants (TCAs) and/or benzodiazepines (BZDs). This is a potentially dangerous drug combination. The authors hypothesized that the presence of methadone and a TCA, a BZD, or both is associated with an "accidental" overdose (AOD) death more often than a death from any other cause., Methods: A retrospective chart review of New York City Office of Chief Medical Examiner data for 2003 was performed. Decedents who tested positive for methadone that were classified as an AOD death, as determined by the medical examiner, were compared with deaths from all other causes for the presence of a TCA, a BZD, or both. A logistical regression was performed to develop a multivariate model identifying additional variables associated with a methadone-positive AOD death. A p-value of <0.05 was considered significant, and 95% confidence intervals (CIs) were calculated., Results: In 2003, there were 5,817 medical examiner cases, of which 500 (8.6%) were methadone positive. Of the methadone-positive cases, 493 were available for analysis; 95 (19.3%) were TCA positive and 158 (32.0%) were BZD positive. The odds of having an AOD death in methadone-positive decedents testing TCA positive, BZD positive, or both were 2.11 (95% CI = 1.32 to 3.37; p < 0.01) for TCAs, 1.66 (95% CI = 1.12 to 2.45; p < 0.02) for BZDs, and 4.34 (95% CI = 1.97 to 9.56; p < 0.001) for both. The multivariate logistic regression of analytes revealed the following covariates associated with an AOD death as well: amitriptyline, cocaine, morphine, or opiates., Conclusions: Among the methadone-positive cases, testing positive for a TCA, a BZD, or both was associated with an AOD death.

The impact of P-glycoprotein (P-gp) on the distribution of nortriptyline (NT) and its metabolites between brain, liver and serum was studied experimentally. The interaction of NT with P-gp in vitro was confirmed by measurement of P-gp stimulated ATPase activity (Km = 257.6 microM, Vmax = 51.0 nmol phosphate released/mg protein-min). Administration of NT (5 mg/kg, s.c.) to mdrla knockout mice resulted in enhanced brain-serum (1.6-fold, p = 0.012) and liver-serum (1.4-fold, p = 0.019) ratios, as compared to the wild-type mice. For a series of NT doses (2.5, 5, 10, 25, 30 mg/kg, i.p.) inhibition of P-gp with cyclosporine A (CsA, 200 mg/kg, i.p.) in rats led to NT brain- and liver-serum ratios that were on average 1.3- (p = 0.005) and 2.1- (p = 0.001) fold higher than those of the controls, respectively. Verapamil (50 mg/kg) (NT, 5 mg/kg) increased the ratios by a factor of 1.6 (p <0.001) and 10.3 (p <0.001) for brain and liver, respectively. Finally, co-administration of methadone (1 mg/kg) did not alter the brain-serum ratio of NT, but in the liver a slight increase (1.5-fold, p = 0.035) was observed. In conclusion, verapamil yielded complete inhibition of P-gp at the blood-brain barrier and CsA had an effect corresponding to about 50% inhibition. The results show that P-gp influences the penetration of NT into the brain, and that drug-drug interactions may take place.

Toxicological investigations of postmortem specimens of a 26-year-old man were performed with the use of LC/APCI/MS. They revealed in the blood of the deceased clomipramine (9.49 microg/g) and its main metabolite norclomipramine (1.10 microg/g) at concentrations explaining the fatal outcome. The presence of these xenobiotics in a 12-cm-long strand of hair (clomipramine, 7.60 ng/mg in I segment; 4.19 ng/mg in II segment; 1.86 ng/mg in III segment; norclomipramine, 5.71 ng/mg in I segment; 9.71 ng/mg in II segment; 4.13 ng/mg in III segment) confirmed the fact obtained from the medical history that the deceased had been receiving clomipramine as an antidepressant for 1 year prior to his death. The analysis demonstrated ethanol in autopsy blood (2.5mg/ml) and urine (3.2mg/ml); ethyl glucuronide as a marker of chronic alcohol abuse was detected in the deceased's hair (0.44 ng/mg in I segment; 0.07 ng/mg in II segment; n.d. in III segment). These findings may suggest the contribution of alcohol in the mechanism of drug-ethanol interaction, which in consequence might have affected the biotransformation of clomipramine in the final period of his life and evoked the ultimate toxic effect.

An isocratic reversed-phase liquid chromatographic method is described using a hybrid column for the control of synthetic impurities potentially found in amitriptyline hydrochloride. The method has been validated and is capable of controlling impurities to 0.1 per cent.

Although tricyclic antidepressants have been in existence since the 1940s when they were discovered upon screening iminodibenzyl derivatives for other potential therapeutic uses, their mechanism of action has remained unclear [A. Goodman Gilman, T.W. Rall, A.S. Nies, P. Taylor, Goodman and Gilman's The Pharmacological Basis of Therapeutics, eighth ed., Pergamon Press, New York, 1990]. In addition to their ability to hinder the reuptake of biogenic amines, there is mounting evidence that the tricyclic antidepressants inhibit glutamate transmission. Here, intrinsic tryptophan fluorescence spectroscopy is used to document the binding of desipramine, a member of the tricyclic antidepressant family, to a well-defined extracellular glutamate binding domain (S1S2) of the GluR2 subunit of the amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptor. The binding is distinct from those of other known effectors of the receptor, including the endogenous sulfated neurosteroids pregnenolone sulfate and 3alpha-hydroxy-5beta-pregnan-20-one sulfate, and is consistent with a conformational change upon binding that is allosterically transmitted to the channel region of the receptor.

Scientific interest in saliva as a diagnostic matrix has greatly increased over the last decade. The Triage screening test (Biosite Diagnostics), a rapid immunological test used to detect recreational drugs in the urine, was used to compare two biological matrixes: a non-conventional one, saliva, and a traditional one, urine. Twenty-one drug abusers collected one urine and one saliva specimen, both of which were tested with the Triage kit. Data were validated by gas-chromatography-mass-spectrometry (GC-MS). Results were positive for methadone in 9 saliva and 14 urine specimens, for opiates in 2 and 10, respectively, and for barbiturates in 2 specimens. Saliva specimens were negative for cannabis, THC, benzodiazepines and tricyclic antidepressants, although the GC-MS analysis revealed low concentrations of these drugs in the saliva. The study demonstrates the possibility of using saliva as a diagnostic matrix to test for drug-taking; however, the Triage kit must be improved before being used with saliva.

2,2'-bipyridine is proposed as new and sensitive spectrophotometric reagent for the determination of certain dibenzazepine class of tricyclic antidepressants. The spectrophotometric method is based on the reaction of imipramine hydrochloride (IPH), desipramine hydrochloride (DPH), clomipramine hydrochloride (CPH), trimipramine maleate (TPM) and opipramol (OPP) with iron (III) and subsequent reaction with 2,2'-bipyridine in an acetic acid medium to yield a pink color with maximum absorption at 530 nm. The color developed was stable over 3-4 h at room temperature (approximately 27 degrees C). The commonly encountered excipients and additives did not interfere with the determination. Results from the analysis of pure drugs and commercial tablets agreed well with those of the official method (United States Pharmacopoeia, 24, USP Convention, Rockville 2000, pp. 505-506, 865-867.).

Two simple, precise, and accurate extractive spectrophotometric methods have been developed for the determination of doxepin hydrochloride in pharmaceutical preparations. The methods are based on the formation of ion association complexes of doxepin with titanium (IV) and iron (III) thiocyanate complexes in acidic medium. The produced compounds are insoluble in water but well soluble in some organic solvents. They are extracted with mixtures of butyl alcohol-chloroform (2:3, v/v) and (1:4, v/v) and measured spectrophotometrically at 400 and 490 nm for DOX-Ti-SCN and DOX-Fe(III)-SCN methods, respectively. Beer's law was obeyed in the concentration ranges of 5-50 and 3-30 microg/ml with molar absorptivity of 7.12 x 10(3) and 1.36 x 10(4) l mol(-1) cm(-1) for DOX-Ti-SCN and DOX-Fe-SCN systems, respectively. The proposed methods have been successfully applied for the analysis of the drug in dosage forms. No interference was observed from common pharmaceutical adjuvants. The methods have been also used for the determination of the drug in the presence of its degradation product. Statistical comparison of the obtained results with the reference methods shows excellent agreement and indicates no significant difference in accuracy and precision.

Binding of the tricyclic antidepressant imipramine (IMI) to neutral and negatively charged lipid membranes was investigated using a radioligand binding assay combined with centrifugation or filtration. Lipid bilayers were composed of brain phosphatidylcholine (PC) and phosphatidylserine (PS). IMI binding isotherms were measured up to IMI concentration of 0.5 mmol/l. Due to electrostatic attraction, binding between the positively charged IMI and the negatively charged surfaces of PS membranes was augmented compared to binding to neutral PC membranes. After correction for electrostatic effects by means of the Gouy-Chapman theory, the binding isotherms were described both by surface partition coefficients and by binding parameters (association constants and binding capacities). It was confirmed that binding of IMI to model membranes is strongly affected by negatively charged phospholipids and that the binding is heterogeneous; in fact, weak surface adsorption and incorporation of the drug into the hydrophobic core of lipid bilayer can be seen and characterized. These results support the hypothesis suggesting that the lipid part of biological membranes plays a role in the mechanism of antidepressant action.

The high incidence of psychiatric illness in the postpartum period and the increasing percentage of women who breastfeed has focused attention on the treatment of breastfeeding women with psychotropic medications and, additionally, the exposure of nursing infants to these medications. Consequently, there has been an increased effort to develop standardized methods for quantifying psychotropic medications in breast milk. This paper details a novel method for quantifying the concentrations of multiple selective serotonin reuptake inhibitors (SSRIs) and tricyclic antidepressants (TCAs) in breast milk. The method consists of a common liquid/liquid and solid-phase extraction followed by HPLC separation on a common column and UV detection. Assay system 1 measures fluoxetine, norfluoxetine, fluvoxamine, and paroxetine; assay 2 measures sertraline and desmethylsertraline; and assay 3 measures the TCAs including doxepin, nordoxepin, desipramine, imipramine, nortriptyline, and amitriptyline. The method is shown to be a highly accurate and precise technique for measuring 12 different antidepressants in human breast milk and to be free of the matrix effects often encountered in breast milk drug analyses.

We present the validation of a method using non-aqueous capillary electrophoresis (NACE) for quantitative analysis of four tricyclic antidepressants (TADs) in pharmaceutical formulations and plasma. The method presented high resolution allowing the separation of the TADs in 4.3 min at optimized conditions: 50 mM ammonium acetate, 1 M acetic acid in acetonitrile, capillary with 48 cm in length, 40 cm to the detector, and voltage of 30 kV. Acceptable precision (relative standard deviation R.S.D.14.1% from plasma samples) and linearity were achieved using the internal standard (IS) method. The limits of quantification determined for plasma, after liquid-liquid extraction (LLE), were between 30 and 50 ng ml-1. These values are beyond the plasmatic therapeutic concentration. Our results were found comparable or better than those described in the literature for high performance liquid chromatography (HPLC)-based methods.

Two simple and sensitive kinetic methods for the determination of dothiepin hydrochloride are described. The first method is based on kinetic investigation of the oxidation reaction of the drug with alkaline potassium permanganate at room temperature for a fixed time of 25 min. The absorbance of the colored manganate ions is measured at 610 nm. The second method is based on the reaction of dothiepin hydrochloride with 4-chloro-7-nitrobenzofurazan (NBD-Cl) in the presence of 0.1 mol L(-1) sodium bicarbonate. Spectrophotometric measurement was achieved by recording the absorbance at 470 nm for a fixed time of 60 min. All variables affecting the development of the color were investigated and the conditions were optimized. Plots of absorbance against concentration in both procedures were rectilinear over the ranges 4-24 and 50-250 microg mL(-1), with mean recoveries 99.33+/-0.42 and 99.88+/-0.53, respectively. The proposed methods were successfully applied for the determination of dothiepin hydrochloride in bulk powder and in capsule dosage form. The results obtained were found to agree statistically with those given by the non-aqueous B.P. method. Furthermore the methods were validated according to USP guidelines and also assessed by applying the standard addition technique. The determination of dothiepin hydrochloride by the fixed concentration method is feasible with the calibration equations obtained, but the fixed time method proves to be more applicable.

Case Report: The authors report a case of pediatric cyproheptadine toxicity, initially misdiagnosed as tricyclic toxicity based on the results of a preliminary rapid toxicological serum screen. Although such cross-reactivity has been reported, the chemical basis of this observation has not yet been evaluated. By GC/MS methods and HPLC assays adapted for the detection of tricyclic compounds, the authors confirmed that cyproheptadine was indeed responsible for this patient's toxicity. In addition, the authors identified the presence of a cyproheptadine metabolite in the patient's serum. Further testing in an immunoassay-based toxicologic screen demonstrated some cross-reactivity exhibited by the patient's serum, but not the parent compound. These findings showed that the cross-reactivity correlated with the presence of the cyproheptadine metabolite, highlighting the value of confirmatory toxicologic testing of routine rapid toxicologic screens.

The chromatographic behaviour (retention, selectivity, peak shape and resolution) of seven tricyclic antidepressants (TCAs), amitryptiline, clomipramine, doxepin, imipramine, maprotiline, nortryptiline and trimipramine, was examined. Conventional unendcapped Cs and C18 columns and an endcapped XTerra MS C18 column recommended for the analysis of basic compounds were used together with acetonitrile-water and micellar sodium dodecylsulfate (SDS)-pentanol mobile phases. The two best combinations were XTerra C18/acetonitrile, which yielded the largest efficiencies and resolution, and C8/SDS-pentanol, which eliminated the peak tails that were still observed with the XTerra C18 column. Both the systems were used to develop simple chromatographic procedures for the control of TCAs in pharmaceutical formulations using UV detection. The selected mobile phase compositions were 35% (v/v) acetonitrile (XTerra C18 column) and 0.075 M SDS-6% (v/v) pentanol (C8 column), both at pH 3. Satisfactory recoveries were achieved in both cases, with intra- and inter-day relative standard deviations (RSDs) always below 0.6 and 2.0%, respectively. The preparation of the samples was simple in both modes, since a previous extraction of the drugs was not needed. The micellar mode has, however, the advantage of using a smaller amount of organic solvent, which is retained in the micellar SDS solution. The C8 column is also less expensive.

The effect of variables such as shape template size, porogen composition and percentage, content of cross-linking monomer, and polymerization temperature on the properties of uniformly sized 3-microm porous poly(glycidyl methacrylate-co-ethylene dimethacrylate) beads prepared by the staged templated suspension polymerization technique has been studied. The porous properties of the beads including surface morphology, pore size distribution, and specific surface area have been optimized to obtain highly efficient stationary phases for normal-phase HPLC. A column packed with diol stationary phase obtained by hydrolysis of poly(glycidyl methacrylate-co-ethylene dimethacrylate) beads affords an efficiency of 67,000 plates/m for toluene using THF as the mobile phase. The retention properties and selectivity of the diol beads are easily modulated by changes in the composition of the mobile phase. The performance of these beads is demonstrated with the separations of a variety of polar compounds including positional isomers, aniline derivatives, and basic tricyclic antidepressant drugs.

Polymer-coated fibrous material has been introduced as the extraction medium for a miniaturized sample preparation method being coupled with microcolumn liquid chromatography. The preconcentration and the subsequent liquid chromatographic separation of tricyclic antidepressants (TCAs) drugs, amitriptyline, imipramine, nortriptyline and desipramine, was carried out with the hyphenated system. Several basic experimental parameters, such as extraction and separation conditions, were investigated along with the applicability of the method for the analysis of biological fluids. The results clearly showed that the on-line coupled system could be a powerful tool for the analysis of complex mixtures in biological matrix without a large solvent consumption and specially designed instruments. The lowest limit of quantification was quite acceptable for the analysis of TCAs in clinical and forensic situations.

The problem of acute intoxication has become very urgent now due to a great number of various chemical preparations accumulated during the last decades in the environment. Intoxications with psychotropic drugs and their mixtures form the significant part of the intoxications; there is an increasing tendency of intoxication with several preparations at a time. Amitriptyline and codeine are the preparations, which more frequently can cause intoxication. Fluoxetine is one of the newest and often used antidepressants. Under certain circumstances, like overdose, using all preparations together, long term using or using for suicide, these preparations can be even a cause of death. In such cases amitriptyline, fluoxetine and codeine become the objects of chemical-toxicological analysis. The possibility of the separation and identification of amitriptyline, fluoxetine and codeine in the mixture using thin-layer chromatography was established. Dragendorf reagent, modified by Munje, is most suitable for the spray-distinct of the chromatographic plates for all three substances. Amitriptyline research limit, using this developer, is 0.4 microg, fluoxetine--1.6 microg, codeine--0.8 microg. Most acceptable for separation the components of the mixture are 5 mobile phases: 1. Diethyl acetate-methanol-ammonium hydroxide (concentrated solution) (85:10:5). Amitriptyline, fluoxetine and codeine R(f) medium values respectively are 0.94; 0.63; 0.51. 2. Buthylacetate-methanol-ammonium hydroxide (concentrated solution) (85:10:5). Amitriptyline, fluoxetine and codeine R(f) medium values respectively are 0.65; 0.24; 0.15. 3. Cyclohexane-diethyl acetate-diethyl amine (70:15:15). Amitriptyline, fluoxetine and codeine R(f) medium values respectively are 0.93; 0.75; 0.37. 4. Cyclohexane-buthylacetate-diethyl amine (70:15:15). Amitriptyline, fluoxetine and codeine R(f) medium values respectively are 0.92; 0.51; 0.25. 5. Acetone-1,4-dioxane-ammonium hydroxide (concentrated solution) (30:68:2). Amitriptyline, fluoxetine and codeine R(f) medium values respectively are 0.82; 0.62; 0.42. Recommended methodology for the separation and identification of amitriptyline, fluoxetine and codeine in the mixture using thin-layer chromatography is statistically reliable: when confidence level is 0.95, relative error is less than 0.05; standard deviation is from 0.007 to 0.03. Recommended methodology suits for mixture, extracted from biological liquids, components separation and identification.

A small-scale, homogeneous, rapid sensing system for phenothiazines and tricyclic antidepressants (TCAs) has been developed by employing fluorescently labeled mutant calmodulin (CaM) as the recognition element. A calmodulin mutant containing a unique cysteine residue at position 109 on the protein was expressed in Escherichia coli. Following purification, the environment-sensitive, thiol-specific fluorophores N-[2-(1-maleimidyl)ethyl]-7-(diethylamino)coumarin-3-carboxamide (MDCC), 6-acryloyl-2-dimethylaminonaphthalene (acrylodan), and 4-[N-(2-(iodoacetoxy)ethyl)-N-methylamino]-7-nitrobenz-2-oxa-1,3-diazole (IANBD ester) were coupled to the C109 site of the mutant protein. The response of labeled CaM in the presence of calcium to increasing concentrations of chlorpromazine hydrochloride (CPZ), as well as other phenothiazines and structurally related antipsychotics and antidepressants, was investigated. Fluorescence measurements were performed on benchtop and microtiter plate fluorometers. The responses were characterized as a change in the signal intensity of the labeled protein upon ligand binding, and the stability of the system was monitored over a nine-month period. The assay showed specificity for the phenothiazine and TCA classes of drugs, with limits of detection in the micromolar range. Selectivity studies indicated negligible response of the biosensing system to structurally unrelated compounds. This work represents a proof-of-concept assay for rapid, homogeneous detection of drugs employing binding proteins as the biorecognition element.

Five tricyclic antidepressants (TADs), desipramyne, nortriptyline, imipramine, doxepin and amitriptyline, were separated by using the N,N,N',N'-tetramethyl-1,3-butanediamine (TMBD) as additive in the background electrolyte solution. Because the tricyclic antidepressants are similar in structure, mass and pka values, their separation, by capillary zone electrophoresis, requires the careful manipulation of parameters, such as the pH and the composition of the electrolyte solution. As basic drugs, the TADs interact with the silanol groups on the capillary wall giving rise to peak broadening and asymmetry, non reproducible migration times and failing in selectivity. Different concentrations of TMBD (40, 60, 100 and 150 mM) were used at pH 9.5, but only a 100 mM TMBD allowed a good separation and a high efficiency for all the TADs. At this pH the separation was not possible without additive. This result is due to the reduced electroosmotic flow whose mobility is at a value of 10(-9) m(2) V(-1) s(-1).

A reversed-phase HPLC method with UV detection at 252 nm is presented for the simultaneous determination of some tricyclic antidepressants (amitriptyline, imipramine) and neuroleptics (chlorprothixene, thioridazine) in their quaternary mixtures. Sample analysis was performed on a bonded reversed phase C-18, 5 microm, 250 x 4.6 mm ID (Lichrospher 100RP-18) column using acetonitrile and 0.01 M aqueous solution of triethylamine (1:1) as the mobile phase at 0.9 ml/min. The pH was adjusted to 2.7 with concentrated phosphoric acid. The retention time was for imipramine, amitriptyline, chlorprothixene, and thioridazine 5.8, 6.5, 8.3, 10.8 min, respectively. The linearity was obeyed up to 15 ppm for imipramine and amitriptyline, 12 ppm for chlorprothixene and 10 ppm for thioridazine. The presented method also allows the determination of the mentioned drugs individually in their pharmaceutical preparations.

The qualitative and quantitative method of determination of amitryptilin, codeine and fluoxetine in the mixture using high-pressure liquid chromatography is described in this paper. Chromatogram is presented which shows, that preparations are fully separated and do not interfere each others analysis. Tables with chromatographical separation characteristics are also presented. Proposed calibration curves of quantitative analysis and calculated medium relative error of quantitative ascertainment for every preparation of the mixture are shown. Final conclusion: method is applicable for qualitative and quantitative analysis of amitryptiline, fluoxetine and codeine in the mixture in hasty poisoning cases.

Dialysis-solid-phase extraction (SPE) sample pretreatment is combined on-line with non-aqueous capillary electrophoresis for the determination of tricyclic antidepressants in urine and serum. After clean-up and enrichment, the water is removed from the sample matrix and the analytes are eluted from the cartridge by means of an organic solvent. Next, the eluate is transported to the capillary and the injection is performed electrokinetically. This injection, which does not suffer from an adverse sample matrix effect because of the SPE step, results in further analyte concentration. The detection limits are in the 0.02-0.1 microg/ml range and the day-to-day repeatabilities are between 2.5 and 9.5%, which is quite satisfactory.

Four cation-exchange materials, possessing propanesulfonic acid ligands, for use in capillary electrochromatography were prepared from different commercially available 5-microm bare-silica particles ranging from 80 to 800 A in pore size. The performance of the materials was investigated at different compositions of the mobile phase (pH, ionic strength, and acetonitrile content) using tricyclic antidepressants and related quaternary ammonium analogues as test analytes. The wide-pore materials promoted pore flow, but this had no positive influence on the performance. The small-pore (highest surface area) particles gave, as could be expected, the best selectivity.

Autopsies and toxicological analyses at the Institute of Forensic Medicine revealed 85 fatal intoxications with neuroleptics in Vienna from 1991 to 1997. A total of 17 cases were linked to a single neuroleptic (NL) alone, while 68 deaths were attributed to a combination of NLs with other drugs. The most frequently detected agent was prothipendyl (n=41). During the study period the number of defined daily doses of high-potency NLs prescribed increased significantly (P< or =0.001) due to increased prescribing of new atypical antipsychotics. The quantity of intermediate- and low-potency NLs dispensed remained stable. The most frequently prescribed NL was haloperidol. The relative toxicities of different NLs were calculated by dividing the number of deaths caused by this NL into the number of defined daily doses prescribed in the observation period (f-value). Single-substance intoxications and multiple-substance intoxications were distinguished. The highest f-values were associated with low-potency NLs, especially with prothipendyl, chlorprothixene and levomepromazine. Low f-values were found for the group of high-potency NLs, including flupentixol, fluphenazine, haloperidol and pimozide, as well as olanzapine. Compared to the f-values for all NLs prescribed, f-values for low-potency NLs were shown to be significantly higher concerning single-substance intoxications (P< or = 0.05) and multiple-substance intoxications (P < or = 0.001), while f-values for high-potency NLs were significantly lower (P< or = 0.05 and P< or = 0.001). We are not aware of the psychiatric diagnoses in our post-mortem sample. However, the present results indicate that careless use of low-potent NLs should be avoided in patients with a potential risk of accidental or suicidal overdose.

Mirtazapine, 1, 2, 3, 4, 10, 14b-hexahydro-2-methyl-pyrazino [2, 1-a] pyrido [2, 3-c] [G.L. Stimmel, J.A. Dopheide and S.M. Stahl, Pharmacotheraphy 17(1) (1997) 10] benzazepine, is a new and well tolerated antidepressant. It blocks pre-synaptic alpha2-adrenergic receptors and postsynaptic serotonin type 2 and type 3 receptors. The drug is rapid and completely absorbed after oral administration. Mirtazapine was analyzed by HPLC and gas chromatography with nitrogen-sensitive detection. In this study, mirtazapine was analyzed by using UV spectrophotometry, first and second order derivative spectrophotometry. The type of solvent, the degree of derivation, range of wavelength and n value were chosen in order to optimize the conditions. The concentration of mirtazapine in its methanolic solutions were determined between the wavelength range of 225-360 nm in the linearity range of 1-100, 2-100 and 1-120 microg ml(-1) by using the values obtained from UV. first-order derivative (n = 5, delta lambda = 17.5 nm) and second-order derivative (n = 9, delta lambda = 31.5 nm) spectrum of the substance, respectively. The developed UV Spectrophotometric, first-order and second-order derivative spectrophotometric methods were applied to a pharmaceutical preparation as tablet form. Developed UV and derivative UV spectrophotometric method in this study are accurate, sensitive, precise, reproducible and can be directly and easily applied to the pharmaceutical preparations.

Three simple and selective methods are proposed for the determination of nortriptyline hydrochloride in bulk form and in tablets. The first two methods are based on the formation of charge-transfer complexes between the drug base as a n-donor and quinhydrone or p-chloranil as pi-acceptor. The products exhibit absorption maxima at 497 and 560 nm in acetonitrile for quinhydrone and p-chloranil, respectively. The third method is based on the interaction of N-alkylvinylamine formed from the condensation of the free secondary amine group and acetaldehyde with p-chloranil to give a vinylamino substituted quinone. The coloured product exhibits an absorption maximum at 650 nm in dioxane. All variables were studied to optimize reaction conditions. Beer's law was obeyed and the relative standard deviations were found to be less than 1.5%. The methods have been applied to the analysis of nortriptyline hydrochloride in the bulk drug and in tablets.

Eriochrome cyanine R (ECR) has been tested as reagent for the determination of imipramine. It reacts in neutral medium with imipramine forming reddish compound, which can be quantitatively extracted into n-butanol. This property has been successfully used for the extractive-spectrophotometric determination of imipramine. Beer's law is obeyed in concentration range of 10-80 microg ml(-1) of imipramine. The method was applied to the determination of imipramine in its pharmaceutical.

An ingestion of an unknown quantity of Harmomed (dothiepin and diazepam) capsules in a suicide is described. The authors report a new and fast method of analysing and determining the dothiepin concentration in postmortem specimens. Quantitation of dothiepin, and its metabolite desmethyldothiepin was performed by ethyl acetate extraction from alkalinized body fluids before GC-MS analysis. The analyses were performed without any complex sample clean-up steps and with little sample material. Postmortem concentrations of dothiepin, desmethyldothiepin, diazepam and desmethyldiazepam in body fluids are given. The proposed method is a rapid procedure for analysis in cases of deliberate poisoning with the antidepressant drug dothiepin.

A new rapid, reliable and specific UV spectrophotometric method was developed for the determination of nortriptyline hydrochloride formulated into o/w microemulsions. The UV spectra of nortriptyline standard solution in methanol and placebo (microemulsion without nortriptyline) were recorded over the wavelength range 200-600 nm and the spectra for placebo and nortriptyline loaded microemulsion were recorded over the range 260-400 nm in order to determine the overlapping that might appears, and hence to set the wavelength that could be used for the quantitative analysis. This method was validated and compared with a liquid chromatography (LC) procedure used for the quantitative analysis of the drug. Both methods showed excellent precision and accuracy with RSD values of 2.37 and 1.41%, respectively, for the LC method, and values of 1.24 and 2.88%, respectively, for the UV spectrophotometric method. The established linearity range was 10-50 microg ml(-1) (r2 = 0.9985) and 20-60 microg ml(-1) (r2 = 0.9979) for the HPLC and UV spectrophotometric methods respectively. The recoveries of nortriptyline from spiked placebos were > 95% for both methods over the linear range. The methods have been successfully used for determining the nortriptyline content of microemulsions and for evaluating the chemical stability of the drug in nortriptyline-loaded microemulsions.

Stationary phases were investigated for HPLC coupled with electrospray ionization mass spectrometry (ESI-MS) for the analysis of basic drugs. Tricyclic antidepressants (TCAs) and beta-blockers were used as model solutes. The functional groups, pentafluorophenyl (PFP), OH, CN or CH3 were attached to the silica via a propyl chain. The effects of these stationary phases as well as C8 and C18 phases on retention and peak shape of the basic drugs were studied. The CN and PFP phases adequately retained (tR of 2 to 6 min) the basic drugs when the mobile phase was composed of 90% acetonitrile, whereas with the C4, C8 and C18 phases, less than 40% acetonitrile had to be used to provide adequate retention of the basic drugs. Because acetonitrile provides better desolvation in ESI than an aqueous solvent, it produces an increased MS signal. As an example of the HPLC-ESI-MS analysis of the beta-blocker, pindolol, on a CN phase, the use of 90% acetonitrile in the mobile phase increased the ESI-MS signal by 790% when compared to a C18 phase which could use only 5% acetonitrile in the mobile phase for retention of the solute. In addition, the CN and PFP phases provided better peak shape than the OH phase and the hydrophobic phases (C4, C8 and C18) and ion-pairing or ion-suppressing agents were not required. The retention behavior of the TCAs and beta-blockers on each of the phases is described.

Simple and sensitive method for determination of imipramine and desipramine is reported. The procedure is based on the oxidation of the drugs by ammonium metavanadate. Linear calibration graphs were obtained in the concentration range 0.6-40 microg ml(-1) of imipramine and 0.7-35 microg ml(-1) of desipramine with a relative standard deviation (RSD) less than 0.5%. The method was applied to the determination of the drugs in pharmaceutical preparations and compared favourably with independent official methods.

Headspace solid phase microextraction (HS-SPME) has advantages of high purity of the extract, avoidance of organic solvents and simple technical manipulation and can be used in combination with gas chromatography-mass spectrometry (GC-MS) in the hair analysis of a number of drugs. HS-SPME coupled with the hydrolysis of the hair matrix by 4% sodium hydroxide in the presence of excess sodium sulphate and of a suitable internal standard proved to be a convenient one-step method for the measurement of many lipophilic basic drugs such as nicotine, amphetamine derivatives, local anaesthetics, phencyclidine, ketamine, methadone, diphenhydramine, tramadol, tricyclic antidepressants and phenothiazines. Detection limits were between 0.05 and 1.0 ng/mg. From spiked 10-mg hair samples absolute recoveries between 0.04 and 5.7% were found. These recoveries decreased considerably if larger sample amounts were used, perhaps due to increased drug solubility in the aqueous phase or to elevated viscosity in the presence of dissolved hair proteins. Because of the phenolic hydroxyl group a change of pH after alkaline hair digestion (by adding excess orthophosphoric acid) was necessary for the detection of delta 9-tetrahydrocannabinol (delta 9-THC), cannabinol (CBN) and cannabidiol (CBD) by HS-SPME. Nevertheless, the detection limits were such that only CBN could be detected in hair of a consumer. Clomethiazole, a compound hydrolysed in alkali, was measured by HS-SPME after extraction with aqueous buffer. The detection limit was 0.5 ng/mg. Cocaine could not be detected by HS-SPME. The application of HS-SPME to hair samples from several forensic and clinical cases is described.