Development and validation of a sensitive spectrofluorimetric method for the determination of amoxapine in human plasma and urine.

A selective and sensitive spectrofluorimetric method was developed and validated for the determination of amoxapine in human plasma and urine. The developed method is based on labeling with 5-dimethylaminonaphthalene-1-sulfonyl chloride (dansyl chloride) and monitoring at 397 nm (excitation)/514 nm (emission). The method was validated for linearity, limit of detection (LOD), limit of quantification (LOQ), precision, accuracy, recovery and robustness. The calibration curves were linear over a concentration range of 250-2500 and 50-1250 ng/mL for plasma and urine, respectively. The LOD values were calculated to be 13.31 and 13.17 ng/mL for plasma and urine, respectively. The proposed method was applied to study of amoxapine in human plasma and urine.

Investigation of the disposition of loxapine, amoxapine and their hydroxylated metabolites in different brain regions, CSF and plasma of rat by LC-MS/MS.

Loxapine represents an interesting example of old "new" drug and is recently drawing attention for its novel inhalation formulation for the treatment of both psychiatric and non-psychiatric disorders. It is extensively metabolized to several active metabolites with diverging pharmacological properties. To further pursue the contribution of metabolites to the overall outcome after loxapine administration, quantification of both loxapine and its active metabolites is essential. The current study developed a rapid liquid chromatography-tandem mass spectrometry (LC-MS/MS) method for the simultaneous quantification of loxapine and its five metabolites (amoxapine, 7-hydroxy-loxapine, 8-hydroxy-loxapine, 7-hydroxy-amoxapine and 8-hydroxy-amoxapine) in rat brain tissues, plasma and cerebrospinal fluid (CSF). By evaluating the effects of perchloric acid and methanol on analyte recovery, the extraction methods were optimized and only small amounts of sample (100 µl for plasma and less than 100mg for brain tissue) were required. The lower limits of quantification (LLOQs) in brain tissue were 3 ng/g for loxapine and amoxapine and 5 ng/g for the four hydroxylated metabolites of loxapine. The LLOQs were 1 ng/ml for loxapine and amoxapine and 2 ng/ml for the four hydroxylated metabolites in plasma, and 10 ng/ml for all analytes in CSF. The developed method was applied to a pharmacokinetic study on rats treated with a low-dose loxapine by oral administration. Four hours after loxapine dosing, high levels of 7-hydroxy-loxapine were found throughout the ten brain regions examined (68-124 ng/g), while only trace amount of loxapine was measured in brain (<5 ng/g) and plasma (<3 ng/ml). The method provides a useful tool for both preclinical and clinical investigations on the dispositions of loxapine and its metabolites, which would help to elucidate their roles in neurotherapeutics.

Validation of HPLC-MS/MS methods for analysis of loxapine, amoxapine, 7-OH-loxapine, 8-OH-loxapine and loxapine N-oxide in human plasma.

BACKGROUND: Two ESI-LC-MS/MS methods were validated for the quantitative analysis of loxapine, amoxapine, 7-OH-loxapine, 8-OH-loxapine and loxapine N-oxide in human K(2)EDTA plasma. Cation-exchange solid-phase extraction (SPE) was used to extract loxapine, amoxapine and the two hydroxylated metabolites, and organic precipitation was used to quantify loxapine N-oxide. RESULTS: Both methods were shown to be accurate (±13%), intra-assay precision was less than 15%, and inter-assay precision was less than 10% in all instances across the entire dynamic range of the assays (0.0500-50.0 ng/ml for the SPE method and 0.100-25.0 ng/ml for the precipitation method). CONCLUSION: The validated methods for loxapine, amoxapine, 7-OH-loxapine, 8-OH-loxapine and loxapine N-oxide have been used to successfully support clinical trials.

Determination of amoxapine and nortriptyline in blood plasma and serum by salt-assisted liquid-liquid microextraction and high-performance liquid chromatography.

A new, selective and sensitive method has been developed for the determination of tricyclic antidepressant drugs, amoxapine and nortriptyline, in human blood plasma and serum, involving their reaction with allyl isothiocyanate and extraction of thiourea derivatives with water-miscible organic solvent acetonitrile. The phase separation was effected by addition of ammonium sulphate, a process called salt-assisted liquid-liquid microextraction. The extract was analyzed by HPLC with UV detection at 254 nm. The method has been optimized for derivatization reaction time and temperature, solvent for extraction, and salt for solvent phase separation. Under the optimal conditions, a linear calibration graph was obtained between the amount of drug and the peak area of thiourea derivatives in the range of 0.002-20 mg/L drugs. The correlation coefficient and limit of detection values for amoxapine and nortriptyline in serum/plasma samples were in the range of 0.9953-0.9999 and 0.46-0.58 µg/L, respectively. The recovery in spiking experiments ranged, respectively, 75-88% (RSD 3.4-7.2%) and 79-97% (RSD 3.7-7.9%) for the two drugs.

An autopsy case of poisoning with ethanol and psychotropic drugs.

A case of fatal poisoning involving ethanol with psychotropic drugs is presented. Quantitative toxicological analysis showed that the concentrations of ethanol, amoxapine and phenobarbital in the femoral blood were 2.86 mg/ml, 0.41 microg/ml and 6.80 microg/ml, respectively. We concluded that the cause of death was due to the combination use of ethanol, amoxapine and phenobarbital.

Determination of tricyclic antidepressants in human plasma using pipette tip solid-phase extraction and gas chromatography-mass spectrometry.

A method for the simultaneous extraction of four tricyclic antidepressants from human plasma samples using pipette tip SPE with MonoTip C(18) tips is presented. Human plasma (0.1 mL) containing four tricyclic antidepressants (amitriptyline, amoxapine, imipramine, and trimipramine) and an internal standard (IS), protriptyline, was mixed with 0.4 mL of distilled water and 100 microL 1 M NaOH solution. After centrifugation of the mixture, the supernatant was extracted to the C(18) phase of the tip by 20 repeated aspirating/dispensing cycles using a manual micropipettor. The analytes retained in the tip were eluted with methanol by five repeated aspirating/dispensing cycles. Without evaporation and reconstitution, the eluate was directly injected into a gas chromatograph injector and detected by a mass spectrometer with SIM in the positive-ion electron impact mode. Recovery of the four antidepressants and IS spiked into human plasma was 80.2-92.1%. The regression equations for the four antidepressants showed excellent linearity in the range of 0.2-40 ng/0.1 mL. LODs and LOQs for the four drugs were 0.05-0.2 ng/0.1 mL and 0.2-0.5 ng/0.1 mL, respectively. Intra- and interday CVs for the four drugs in plasma were no greater than 9.5%.

The role of metabolites in a bioequivalence study II: amoxapine, 7-hydroxyamoxapine, and 8-hydroxyamoxapine.

BACKGROUND: Amoxapine is a dibenzoxazepine type tricyclic antidepressant. The mechanism of clinical action in patients is not well understood. In animals, amoxapine blocks the reuptake of norepinephrine and, to a lesser extent serotonin, into their respective neurons and blocks the response of dopaminergic receptors to dopamine. The major metabolite, 8-hydroxyamoxapine, has similar norepinephrine uptake inhibitory action to the parent drug, but has a more pronounced inhibitory action on serotonin uptake. Another major metabolite, 7-hydroxyamoxapine blocks post-synaptic dopamine receptors. SUBJECTS AND METHODS: The present study was a traditional two-treatment, two-period, two-sequence crossover design with the primary objective to investigate the average bioequivalence of two formulations of amoxapine. Secondary objectives were to explore the potential roles of metabolites and truncated (partial) areas in bioequivalence studies. Serial plasma samples were harvested from immediately pre dose to 96 hours post dose. The parent drug and the two hydroxy metabolites were monitored by validated HPLC procedures. Geometric mean ratios and 90% confidence intervals (90% CIs) were calculated for Cmax, AUCinfinity, the truncated areas of AUC, AUCinfinity/Cmax, and the truncated areas of AUC/Cmax. RESULTS: The results indicated that the two formulations were bioequivalent in terms of the conventional parameters Cmax and AUC for all three analytes in the sense that the 90% CIs fitted entirely within preset bioequivalence limits of 80-125%. Moreover, the 90% CIs for the truncated areas AUC2.0hr through AUClast and Cmax/AUC1.0hr through Cmax/AUClast of all three analytes also fell entirely within bioequivalence limits of 80-125%. It was concluded that it was unnecessary to have harvested plasma samples beyond 12 hours, in which case additional plasma samples could have been devoted to the more precise estimation of tmax and Cmax. CONCLUSION: Of the three analytes, test and reference individual plasma concentration versus time curves of 8-hydroxyamoxapine were more closely superimposable than those of the other two analytes. Any decision to use metabolite data in bioequivalence studies, however, must be made a priori to avoid introduction of bias arising from selective post hoc manipulation of the raw data; and to facilitate the design of blood sampling schedules based on prior information about the tmax of the selected analyte.

Concurrent high-performance liquid chromatographic measurement of loxapine and amoxapine and of their hydroxylated metabolites in plasma.

The dibenzoxazepine neuroleptic loxapine, its N-demethylated metabolite amoxapine, and their 7- and 8-hydroxymetabolites were measured simultaneously in plasma by reversed-phase high-performance chromatographic method. An original liquid-liquid extraction procedure was performed, consisting in coextraction of the substances together with a water-miscible solvent (acetonitrile) by a non-water-miscible solvent (toluene). The substances were separated on a 5-microm CN 25-cm column, and eluted with a mobile phase consisting of acetonitrile-acetic acid 0.5 N (30:70) and hexylamine (0.05%). They were detected by ultraviolet spectrophotometry at 310 nm. Clozapine was used as internal standard. Linearity was demonstrated in the range of 10 to 250 microg/l, and detection limits were found to be 3.5 to 6.3 microg/l according to the substance. Within-day repeatability ranged from 2.7% to 6.5%, and between-day reproducibility ranged from 0.9% to 20.2%. The extraction procedure provided a mean absolute recovery of 51.1% (range, 40.7% to 58.6%) with a mean coefficient of variation of 4.2%. This technique was applied to the concurrent determination of plasma concentrations of the compounds in 10 patients administered loxapine 75 to 600 mg daily. Steady state plasma levels of loxapine were significantly correlated with oral doses (n = 10, r = 0.858, p < 0.002). In conclusion, the method proved to be a convenient and reproducible procedure allowing the simultaneous measurement of loxapine, amoxapine, and their metabolites in patients.

Simultaneous quantitation of loxapine, amoxapine and their 7- and 8-hydroxy metabolites in plasma by high-performance liquid chromatography.

Loxapine, its N-demethylated metabolite amoxapine, and their 7- and 8-hydroxy metabolites were determined simultaneously in plasma by a simple two-step extraction procedure followed by reversed-phase liquid chromatography. Baseline separation was achieved by a 5-microns Spherisorb C6 column. The mobile phase consisted of 5 mM phosphate buffer (with 14 mM orthophosphoric acid)-acetonitrile (with 105 microM nonylamine) (77:23, v/v). Assays of the steady-state plasma samples obtained from seventeen patients on loxapine showed substantial amounts of 8-hydroxy metabolites, lesser amounts of loxapine, amoxapine and 7-hydroxyloxapine and trace amounts of 7-hydroxyamoxapine. As 8-hydroxy metabolites possess only weak dopamine-D2 blocking activity, the final neuroleptic property of loxapine may be affected significantly by metabolic polymorphism.

Automated determination of drugs in serum by column-switching high-performance liquid chromatography. IV. Separation of tricyclic and tetracyclic antidepressants and their metabolites.

We describe automated column-switching high-performance liquid chromatography for determining nine tricyclic and tetracyclic antidepressants (TCAs) and their metabolites in human serum. TSKgel ODS-80TM and TSKprecolumn PW (Tosoh Co., Tokyo) are used in the analytical column and the precolumn, respectively. A 200-microL serum sample is directly injected onto the precolumn. After washing the serum proteins from the precolumn with potassium phosphate buffer, the precolumn connection is switched to introduce the retained substances onto the analytical column. The drugs are then eluted within 30 min with an acetonitrile/potassium phosphate buffer mixture containing sodium 1-heptanesulfonate. The analytical recoveries (95-104%), reproducibilities (within-run CV less than 3%), and detection limits (10 micrograms/L) indicate that this HPLC system is suited for therapeutic drug monitoring. Correlations were good between the TCA concentrations in serum and administered dose (r = 0.713, n = 41), and between 10-hydroxynortriptyline and nortriptyline in serum (r = 0.691, n = 24).

Determination of amoxapine and its metabolites in human serum by high-performance liquid chromatography.

The simultaneous quantitative determination of amoxapine, 7-hydroxyamoxapine and 8-hydroxyamoxapine in human serum was established, with good recoveries, using reversed-phase high-performance liquid chromatography (HPLC). Prior to analysis by high-performance liquid chromatography, the enzymic hydrolysis with beta-glucuronidase/arylsulphatase of sera from healthy volunteers receiving the drug showed that each conjugate of two hydroxyamoxapines was 75-90% of the amount determined by the present method. The concentrations of amoxapine and its hydroxylated metabolites were measured against time in sera from the volunteers who were given the antidepressant orally for 2 weeks. The serum levels of 8-OH-amoxapine were markedly higher than the drug itself and the 7-OH-derivative. Whereas the levels of the drug were little increased during the continuous administration, the levels of 8-OH-amoxapine were linearly increased until the fourth day after the administration was started. In addition, the ratio of each hydroxylated metabolite to the drug and the time-course of their serum levels varied interindividually.

Pharmacokinetics of amoxapine and its active metabolites.

The plasma concentrations of amoxapine and its active metabolites, 8-hydroxyamoxapine and 7-hydroxyamoxapine were determined in 8 healthy volunteers receiving a single oral dose of 100 mg of the drug. Considerable interindividual variation was seen in the plasma levels of the three substances. Amoxapine reached maximum levels of 67.4 +/- 35.8 ng/ml between 1 and 2 h after administration. The decline of amoxapine levels in plasma was biphasic. The mean elimination half-life was 9.8 +/- 2.6 h and the estimated first-pass loss ranged between 0.18 and 0.54. The peak levels of the metabolites were reached between 1 and 3 h after administration, with 8-hydroxyamoxapine levels significantly higher than those of 7-hydroxyamoxapine. The mean elimination half-lives were 30.8 and 5.1 h for 8-hydroxyamoxapine and 7-hydroxyamoxapine respectively. The margins of the plasma concentrations reached at steady-state were calculated according to pharmacokinetics parameters for a dosage interval of 8 h.

Amoxapine fatalities: three case studies.

Amoxapine (Asendin), a recently introduced dibenzoxazepine, has been effective in clinical studies for the treatment of various types of depression. Three amoxapine-related deaths are reported. Quantitation of amoxapine was carried out by gas chromatography using 3% OV-17 column. Blood amoxapine concentrations were 11.5 mg/l, 2.8 mg/l, and 0.89 mg/l. The concentrations are many-fold higher than the reported therapeutic serum concentrations of 0.21 mg/l. These cases illustrate the potential toxicity and lethality of amoxapine overdose and the need for caution in prescribing a large amount of amoxapine to patients with suicidal tendencies.

Isocratic liquid chromatographic method for the determination of amoxapine and its metabolites.

An isocratic reverse-phase liquid chromatographic method for the determination of amoxapine and its major metabolites in human plasma utilizing UV detection is described. Plasma samples were extracted with ethyl acetate after pH adjustment. The reconstituted extracts were injected onto a cyanopropylsilane column and eluted with a mobile phase consisting of 65% acetonitrile and 35% sodium acetate buffer, 0.03 M and pH 6. The minimum detectable limit was less than 10 ng/mL of plasma. Possible interferences from other drugs which might be administered concurrently were studied. The reproducibility and precision of the method are demonstrated by the analysis of samples containing 25-600 ng/mL of plasma. The method is being applied successfully in our laboratory for the analysis of plasma from patients receiving amoxapine.

Generalized convulsions as the presenting sign of amoxapine intoxication.

Two infants presented for medical evaluation with sudden onset of seizures or coma, without obvious cause. Suspicious circumstances led to toxicological screening analysis. Amoxapine, a recently released antidepressant, was found in the gastric contents of both children an undetermined time after the putative ingestion, but elevated serum concentrations were noted only in one. The pharmacokinetics are described. There were no obvious cardiotoxic or anticholinergic effects in these infants. Thus, they, like older children and adults, manifest mainly central nervous system toxicity rather than the cardiotoxicity and anticholinergic effects of overdose seen with tricyclic antidepressants.

Double-blind comparison of amoxapine and imipramine in the treatment of depressed patients.

A 5-week double-blind study compared amoxapine to imipramine (2:1 dosage ratio) in the treatment of depressed outpatients. The two agents were similar in anti-depressant efficacy and rapidity of action. The most common adverse reactions to both drugs were anticholinergic effects and sedation; cardiovascular effects were minimal. A few amoxapine-treated patients developed adverse effects typical of neuroleptic drugs: some experienced extrapyramidal signs, one developed galactorrhea, and most showed elevated plasma prolactin concentrations. Amoxapine was associated with significant neuroleptic activity in plasma. No correlation was found between blood levels of either drug and therapeutic response.