Repetitive injection field-amplified sample stacking for cationic compounds determination.

The development of a field-amplified sample stacking technique is presented. Sensitivity enhancement in this technique was obtained by repetitive injections of a sample followed by steps of sample matrix removal through the application of counter-pressure. Under optimized conditions the background electrolyte (BGE) was composed of 80 mM H3PO4 while the sample matrix contained 0.5mM H3PO4 and 30% (v/v) methanol. The elaborated method enabled a 4-fold effective injection of the sample (53 s, 0.5 psi). Each injection was followed by a focusing step during which the application of a voltage (2 kV) and counter-pressure (-1 psi) was performed for 0.65 min. The method was developed for the determination of six psychiatric drugs (opipramol, hydroxyzine, promazine, amitriptyline, fluoxetine, and thioridazine). The elaborated method was applied for analysis of human urine samples after a simple liquid-liquid extraction procedure. The detection limits obtained were in the range of 2.23-6.21 ng/mL.

Extraction and determination of some psychotropic drugs in urine samples using dispersive liquid-liquid microextraction followed by high-performance liquid chromatography.

A simple, rapid and sensitive method termed as dispersive liquid-liquid microextraction (DLLME) combined with high-performance liquid chromatography-ultraviolet detector (HPLC-UV) has been proposed for the determination of three psychotropic drugs (amitryptiline, clomipramine and thioridazine) in urine samples. The determination was performed on a C(8) column under the optimal chromatographic conditions (mobile phase: ammonium acetate (0.03 mol L(-1), pH 5.5)-acetonitrile (60:40, v/v); flow rate: 1.0 mL min(-1); detection wavelength: 238 nm). Several factors influencing the extraction efficiency of the target drugs, such as pH, extraction and disperser solvent type and their volume, extraction time and ion strength were studied and optimized. Under the optimal DLLME conditions, the absolute recoveries of amitryptiline, clomipramine and thioridazine from the urine samples were 96, 97 and 101%, respectively. The detection limits (LODs) and quantification (LOQs) of the proposed approach were 3 and 10 ng mL(-1) for amitryptiline, 7 and 21 ng mL(-1) for clomipramine, and 8 and 25 ng mL(-1) for thioridazine, respectively. The relative standard deviations (RSDs) for nine replicate determinations at 0.100 microg mL(-1) level of target drugs were less than 4.8%. Good linear behaviors over the investigated concentration ranges were obtained with the values of R(2)>0.998 for the target drugs. The proposed method was successfully applied to the real urine samples from two female patients under amitryptiline and clomipramine treatment, respectively.

Separation of promethazine and thioridazine using capillary electrophoresis with end-column amperometric detection.

Promethazine and thioridazine were separated and detected by capillary electrophoresis with end-column amperometric detection. The influence of pH value on oxidation potential, the peak current and the resolution were studied and the following conditions was selected: 0.03 M Na2HPO4 and 0.015 M citric acid at pH 3.0, detection potential at 1.10 V. The detection limits of these two substances were in the range of 10(-8) mol/l. The linear range spanned two to three orders of magnitude. This method was applied to the detection of promethazine and thioridazine spiked in urine.

Metabolism of piperidine-type phenothiazine antipsychotic agents. IV. Thioridazine in dog, man and rat.

1. The metabolism of thioridazine was studied in adult male volunteers, female rat and female dog after oral administration of 50 mg, 20 mg/kg and 100 mg over 30 h, respectively. 2. Metabolites in organic extracts of the urine obtained from each species were analysed by plasmaspray h.p.l.c.-mass spectrometry. For phenolic metabolites the crude extracts from each species were derivatized with a silylating reagent (with and without prior enzymic hydrolysis) prior to h.p.l.c.-mass spectrometric analysis. The structures of metabolites, with the exception of phenols, were confirmed by comparison of their chromatographic behaviours and mass spectral data with those of authentic standards. 3. The metabolites identified in the urine of all three species were mesoridazine, sulforidazine, thioridazine ring sulphoxide, mesoridazine ring sulphoxide, sulforidazine ring sulphoxide, the lactam of mesoridazine ring sulphoxide and unconjugated phenolic derivatives of mesoridazine and sulforidazine. Other compounds observed were: unchanged thioridazine (dog, rat), sulforidazine N-oxide (man), N-desmethylthioridazine ring sulphoxide (dog, rat), N-desmethylmesoridazine ring sulphoxide (dog, rat), the lactam of sulforidazine ring sulphoxide (rat, man), phenolic derivative of thioridazine in unconjugated form (rat), and conjugated form (man), and conjugated phenolic derivative of mesoridazine (man). 4. Thioridazine and six of its metabolites present in the urine of man, rat and dog were quantified by a h.p.l.c.-UV procedure. The mean total urinary excretion (+/- SD) of the measured analytes in man, rat and dog were determined to be 4.3 +/- 2.9, 4.8 +/- 1.7 and 12.1 +/- 5.4% of the dose, respectively. The mean excretion of the lactam of mesoridazine ring sulphoxide was greater in man (1.2 +/- 1.0%) and rat (0.2 +/- 0.2%) than dog (< 0.02%). Moreover, the mean excretion of the lactam of sulforidazine ring sulphoxide was quantifiable in both man (0.5 +/- 0.4%) and rat (0.2 +/- 0.2%). 5. Interspecies comparison of the lactam metabolites indicated that both qualitatively and quantitatively, man more closely resembled rat than dog. Similar observations were previously reported for mesoridazine and sulforidazine, therefore rat may be a more suitable animal than dog to undertake further study of the importance of C-oxidation of the piperidine ring of this class of drug.

Light-induced racemization: artifacts in the analysis of the diastereoisomeric pairs of thioridazine 5-sulfoxide in the plasma and urine of patients treated with thioridazine.

The ring sulfoxidation of thioridazine (THD), a widely used neuroleptic agent, yields two diastereoisomeric pairs, fast- and slow-eluting (FE and SE) thioridazine 5-sulfoxide (THD 5-SO). Until now, studies in which concentrations of these metabolites were measured in THD-treated patients have revealed no significant differences in their concentrations. Preliminary experiments in our laboratory had shown that sunlight and, to a lesser extent, dim daylight led to racemization and probably also to photolysis of the diastereoisomeric pairs as measured by high-performance liquid chromatography. Similar results were also obtained with direct UV light (UV lamp). In appropriate light-protected conditions, THD, northioridazine, mesoridazine, sulforidazine, and FE and SE THD 5-SO were measured in 11 patients treated with various doses of THD for at least 1 week. Significantly higher concentrations of the FE stereoisomeric pair were found. The concentration ratios THD 5-SO (FE)/THD 5-SO (SE) ranged from 0.89 to 1.75 in plasma and from 1.15 to 2.05 in urine. Because it is known that the ring sulfoxide contributes to the cardiotoxicity of the drug even more potently than the parent compound does, these results justify further studies to determine whether there is stereoselectivity in the cardiotoxicity of THD 5-SO.

Sulphoxide metabolites of thioridazine in man.

Chromatograms of urine extracts from patients taking thioridazine contain several different sulphoxide metabolites. Some of these have not hitherto been described or identified. Two of the unknown metabolites appear, from chemical tests and mass spectrometry, to be isomers of the already known thioridazine ring-sulphoxide and sulphoridazine ring-sulphoxide from which they can be produced by treatment with trifluoroacetic anhydride. Two others appear, by similar identification tests and i.r. analysis, to be lactams of mesoridazine ring-sulphoxide and of sulphoridazine ring-sulphoxide.

The biotransformation of thioridazine to thioridazine 5-sulfoxide stereoisomers in phenobarbital induced rats.

The disposition and urinary elimination of thioridazine 5-sulfoxide (ring sulfoxide) following subacute administration of thioridazine was investigated in control and phenobarbital (Pb) induced rats. The ring sulfoxide exists as two stereoisomeric pairs, identified by high performance liquid chromatography as fast eluting (RSF) and slow eluting (RSS) ring sulfoxide. No major differences were detected in the distribution in serum or tissue of the ring sulfoxide between control and induced animals. The ring sulfoxide accumulated in all tissues studied. RSF tissue concentrations were significantly greater than those of RSS in both groups. However, stereoselective formation and elimination of the two ring sulfoxides was not noted in either group. Pb inducible cytochrome P-450 drug metabolizing enzymes do not appear to play a major role in the biotransformation of thioridazine to the ring sulfoxide.

Phenolic metabolites of thioridazine in man.

After oral administration of thioridazine or mesoridazine to man, faecal extracts contained mainly unconjugated 7-hydroxy-thioridazine and its demethylated analogue, together with some 3-hydroxy-thioridazine, and 7-hydroxy derivatives of sulphoridazine and desmethyl sulphoridazine. In contrast, urine contained free 7-hydroxy-mesoridazine, and no free 7-hydroxy-thioridazine was detectable. Conjugates of all these phenols were also found. After oral sulphoridazine administration (thioridazine side-chain sulphone), urine contained mainly 7-hydroxy-sulphoridazine and faeces both 7-hydroxy-sulphoridazine and its desmethyl analogue. Sulphoridazine metabolism is therefore much simpler than that of thioridazine. This different elimination pattern in faeces and urine may be due to reduction of sulphoxides but not sulphones by intestinal bacteria.

Measurement of thioridazine in blood and urine.

1 Thioridazine can be specifically, simply, and reliably measured in plasma and urine by gas chromatography using hexane extraction and prochlorperazine as internal standard; fluorimetry is non-specific. 2 The method can also measure thioridazine ring sulphoxide, and mesoridazine-plus-sulphoridazine (M/S). 3 After single doses plasma sometimes shows M/S in addition to thioridazine itself; it always does so on continued treatment. There is great individual variation in both components, and evidence of changes in metabolism during the early weeks. 4 Urinary excretion may be influenced by pH, but between pH 6.0-7.0 about 1% of the daily dose appears in 24 h urine as the following: free thioridazine in microng quantities, M/S and ring sulphoxide each in mg amounts. 5 Patients attain steady state conditions, although plasma levels rise considerably after each dose and settle again in about 10 h. After chronic treatment is stopped to half-life is at about 30 h. 6 Plasma levels cannot be related to therapeutic response when this is slow, as in schizophrenia, but interpretations are complicated by the production of clinically active metabolites, and by plasma protein binding.

Drug default among schizophrenic patients.

This study attempted to isolate the relationship of two variables that operate to influence drug compliance among schizophrenic patients: the patient's attitude toward his disturbance, and the treatment milieu employed during hospitalization. Schizophrenic patients were administered the Rorschach test upon admission to the psychiatric service of a general hospital to determine their perception of their disturbance. Results of this psychological test were used to randomly select 48 patients who were then randomly assigned to two treatment milius: self-medication during hospitalization and as an outpatient, and traditional drug administration during hospitalization and self-medication as an outpatient. Phenothiazine drugs were used, and compliance was determined by urinalysis. Patients who were generally realistic in their perception of their disorder were significantly more compliant during hospitalization and subsequent outpatient treatment. The treatment milieu variable was not a significant influence. Further analysis via a linear regression model confirmed the relationship of several other compliance factors previously reported in the literature. The role of the patient's attitude toward his illness and the importance of personal relationships are considered in terms of the role of the pharmacist in influencing compliance.

Combined ultraviolet-fluorescence detection in high-pressure liquid chromatography of pharmaceuticals.

The use of combined UV-fluorescence detection for the evaluation of incompletely resolved compounds and trace components in the presence of large quantities of major components is described, analysis for thioridazine and some of its oxidation products by high-pressure liquid chromatography being chosen as a practical example. Mesoridazine and the ring oxide of thioridazine have been determined quantitatively with relative standard deviations (n = 6) of 2.0 and 3.6%, respectively, at concentrations below 0.1 mug per injection. Resolution of the two components is difficult and, in this instance, unnecessary. By a similar approach, it was possible to determine the highly fluoresecent sulforidazine at a concentration of 0.4% of the thioridazine with 6.2 mug of thioridazine injected. A relative standard deviation of 5% was attainable at this concentration. Fluorescence detection limits for mesoridazine and sulforidazine at a signal-to-noise ratio of 4:1 are between 5 and 10 ng per injection; this corresponds to about 0.1% of the active substance for the above example.

Absorption and excretion of thioridazine and mesoridazine in man.

Tritium labeled thioridazine and mesoridazine were given to four schizophrenic subjects to determine if differences in reported clinical potency of these two drugs could be explained by different rates of absorption and excretion. Mesoridazine was found to have earlier peak blood levels and lower fecal excretion. However, the blood and fecal differences were too small to be an adequate explanation for the differences in clinical potency suggesting that the rate of metabolic degradation is a more likely explanation for the potency difference. Thioridazine differs from other phenothiazines by containing two sulfur atoms. Thioridazine is metabolized by oxidative demethylation, oxidation at both sulfur atoms to sulfoxides and sulfones and by hydroxylation in the ring followed by glucuronide formation. Monosufoxides, disulfoxide and disufone have been found in the urine and bile of rats after thioridazine administration by inverse isotope dilution analysis. Neither the ring sulfoxide nor the disulfone show significant pharmacological activity, but activity is shown by the side chain monosulfoxide, mesoridazine. In fact it has been postulated that mesoridazine is the active form of thioridazine. Mesoridazine when compared on an equal dose basis to thioridazine is more potent in anti-emotional and hypotensive effects and produces more extrapyramidal symptoms. Since oxidation of the ring sulfur would be expected to decrease potency, it has been theorized that a portion of thioridazine is oxidized within the ring prior to the oxidation of the side chain sulfur atom thus effectively decreasing the potential activity of thioridazine. Thioridazine studies in rats have shown greater excretion in the urine and bile of the side chain sulfoxide than of the ring sulfoxide or of unchanged thioridazine. The difference in potency of these two compounds could alternatively be a result of differences in absorption, reabsorption after biliary excretion or the rate of urinary excretion. The metabolic pathways of mesoridazine in the human are essentially unknown. Because of this, we thought it worthwhile to determine if the difference in potency between thioridazine and mesoridazine is also related to differences in the rate of excretion and absorption. Because phenothiazines are found in extremely low plasma concentrations, we used radioactive compounds to perform this study.