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The cytochrome P450 enzymes (CYP) 2C19 and 2D6 are involved in the metabolism of many psychotropic drugs. Variability in enzyme activity results in variable metabolic capacities, affecting the metabolism of substrates. The metabolic ratio (MR) of drugs metabolized via these enzymes may therefore reflect the enzyme's activity and/or genotype. To serve as an example for different groups of medications, the selective serotonin reuptake inhibitor venlafaxine, the tricyclic antidepressant amitriptyline, and the antipsychotic risperidone were studied to examine a possible correlation between the MRs of these drugs and the CYP2C19 and/or CYP2D6 genotype. For this purpose data from routine genotyping and serum level analysis were used. The relationships between the observed metabolic ratios and CYP2D6 and/or CYP2C19 genotype were characterized using nonparametric statistical analysis. A clear correlation was observed between the CYP2D6 genotype and the metabolic ratio of venlafaxine. Genotyping of individuals with a log(MR) < -0.6 or a log(MR) > 0.2 would include all patients with an aberrant genotype but would result in a reduction of 52% of genotyping reactions. Slow metabolism of amitriptyline is correlated with a log(MR) > 0.4. Genotyping only those subjects with a log(MR) > 0.4 would result in 88% fewer genotyping reactions. For risperidone, genotyping individuals with a log(MR) > 0.4 would include all CYP2D6 poor metabolizers while reducing the number of genotyping reactions by 93%. According to these data, correlations exist between the log(MR) of venlafaxine, amitriptyline, and risperidone and the genotype of the CYP enzymes involved in their metabolism. From the ranges of log(MR) defined here, a high percentage of aberrant metabolizers can be detected even when patients are not routinely genotyped. Thus, the metabolic ratio may serve as an indication of when genotyping should be considered.

Two cases are presented of intentional intoxications with the tricyclic antidepressants (TCAs) nortriptyline (NT) and amitriptyline (AT). The peak plasma concentrations were 2290 microg/L and 2900 microg/L, respectively. The active metabolites E-10-hydroxynortriptyline (EHNT) and Z-10-hydroxynortriptyline (ZHNT) profiles were quite different as monitored for 5 to 10 days after presumed drug intake. In conclusion, these cases illustrate that (1) metabolite formation and elimination after intake of an overdose dose of NT and AT are stereoselective, and (2) NT and EHNT toxicokinetics and toxicodynamics are quite different. It also shows that a patient with a severe TCA overdose can still survive if he or she receives appropriate and quick supportive care, even if the prognostic markers QRS time, coma grade, and serum TCA levels predict poor outcome.

An analytical method for the simultaneous determination of amitriptyline, nortriptyline, imipramine, desipramine, clomipramine, and desmethylclomipramine in human plasma using promazine as internal standard is described. The method is based on a solid-phase extraction procedure using the Bond-Elut TCA columns followed by separation and detection using capillary gas chromatography with a specific nitrogen phosphorous detector (GC/NPD). Using the new extraction procedure, the problem of adsorption losses was overcome, and good recoveries were achieved for all compounds tested (>87%). Furthermore, clean extracts free of chromatographic interferences were obtained. Complete separation of underivatized, tricyclic antidepressant compounds was achieved in <11 minutes with reliable chromatographic performance. The limits of detection ranged from 1.2 to 5.8 microg/l. Calibration curves, showing good linearity, were prepared covering the therapeutic concentrations range expected (20 to 500 microg/l). The interassay precision values (RSD) ranged from 4.5% to 9.8%. It is concluded that extraction of amitriptyline, nortriptyline, imipramine, desipramine, clomipramine, and desmethylclomipramine, with Bond Elut TCA solid-phase columns followed by their detection using GC/NPD provides a sensitive and reproducible method that can be easily automated for immediate and routine analysis of clinical samples.

In this study the effect of the use of lithium heparin containers on the plasma levels of amitriptyline, clomipramine, and their metabolites was investigated. Twenty-five patients (10 men and 15 women, mean age 51.8 +/- 14.9 years) taking either amitriptyline or clomipramine in a daily dosage varying from 50 to 250 mg entered the study after giving informed consent. From each patient, blood samples were taken in a sodium edetate container and in a lithium heparin container in random order. Parent compound levels are equal in sodium edetate and lithium heparin containers. Metabolite levels are 5.6% lower in lithium heparin containers. This difference is not considered clinically relevant.

Methods for the quantitative determination of amitriptyline and nortriptyline by fluorescence polarization immunoassay (FPIA) is described. One immunoassay allows for the accurate quantification of amitriptyline in the presence of nortriptyline while the second immunoassay allows for the accurate quantification of nortriptyline in the presence of amitriptyline.

Therapeutic drug monitoring data for amitriptyline (AT) and nortriptyline (NT) collected during 10 years (total of 4,278 analyses in 2,937 patients) were evaluated to study how other drugs affect the kinetics at steady state. The distribution of the ratio concentration/daily dose (C/D) in patients treated with the antidepressant only was compared with that in patients on different concomitant drugs. Patients on phenothiazines or dextropropoxyphene had a significantly higher mean C/D of NT than controls, both when AT and when NT had been given. The highest values were seen with levomepromazine and thioridazine. On the contrary, the mean C/D of both AT and NT in patients on carbamazepine was about 50% lower than in those treated with the antidepressant only. Benzodiazepines did not affect the steady-state kinetics of AT or NT. Intraindividual comparisons of the ratio C/D in subjects with analyses performed when off and on concomitant drugs corroborate previous results showing that drugs metabolized by the debrisoquine hydroxylase (CYP2D6) inhibit the metabolism of NT and that carbamazepine induces the metabolism of both AT and NT. Modeling of the dose dependency of the NT interactions with levomepromazine, perphenazine, and thioridazine revealed that the ratio C/D was most affected at low doses of the antidepressant and at high doses of the phenothiazine. The distribution of the doses given was the same in patients on monotherapy as in patients with interacting drugs, which means that many patients treated with phenothiazines had concentrations above the therapeutic range and that most patients treated with carbamazepine had subtherapeutic levels. The present study shows that therapeutic drug monitoring may serve as a valuable tool to discover and quantify drug interactions.

An expedient and specific liquid chromatographic method for a concurrent measurement of fluoxetine (FLU), norfluoxetine (NFLU), amitriptyline (AMI), nortriptyline (NTRIP), imipramine (IMI), and desipramine (DES) is described. Using a mixture of acetonitrile:methanol:0.056 M ammonium acetate:1 M ammonium hydroxide (100:10:4.5:2.6, by volume) as mobile phase, the compounds along with doxepin (DOX) (internal standard) were separated on a 10 mu, 8 mm x 10 cm C18 Resolve cartridge in conjunction with radial compression liquid chromatographic module, and were detected in the effluent spectrophotometrically at 220 nm. A hexane:isoamyl alcohol (98:2, by volume) solution was used for extraction of plasma and the drugs were removed from the organic phase with 0.03% phosphoric acid prior to injection. Under these conditions, no interference in the assay was observed, and the retention times of NFLU, DOX, FLU, AMI, IMI, NTRIP, and DES were 7.8, 11.6, 16, 17.8, 20.9, 31, and 35 min, respectively. The assay was highly linear (r > 0.994), and the within- and between-day coefficient of variance was consistently < or = 9.8%. This assay is currently being used to simultaneously measure these drugs in patients and to investigate their steady-state pharmacokinetics when used in combination.

To interpret blood levels of tricyclic antidepressants, we studied the distributions of amitriptyline and nortriptyline in human blood and explored their control by plasma factors. Each compound (300 ng/ml) was added to whole adult blood and to cord blood with decreased alpha-1-glycoprotein (AGP). Drugs (250 ng/ml) were also added to washed erythrocytes (RBCs) resuspended in autologous plasma or saline (hematocrit = 0.4) with or without AGP, albumin, or tris(2-butoxyethyl) phosphate (TBEP), used to displace AGP-bound drugs. Plasma AGP was determined in all adult blood donors (n = 17). With adult blood, plasma amitriptyline was 393 +/- 52 ng/ml, RBC amitriptyline was 184 +/- 33 ng/ml. Plasma and RBC nortriptyline were 199 +/- 28 and 288 +/- 39 ng/ml, respectively. With saline, cellular amitriptyline and nortriptyline were 81 +/- 10 and 88 +/- 6%, respectively. With plasma, cellular amitriptyline and nortriptyline were 25 +/- 8 and 49 +/- 10%, respectively. The corresponding cord blood values were 52 +/- 12 and 62 +/- 6%. Graded increments of AGP in saline reproduced the distribution pattern seen with increasing concentrations of plasma. Albumin did not influence drug distribution. TBEP markedly increased erythrocyte amitriptyline in adult but not in cord blood. Plasma AGP correlated positively (p = 0.031) with the RBC/plasma ratio of amitriptyline. Amitriptyline is predominantly distributed in plasma, nortriptyline in RBCs. This differential distribution is dose dependent and reflects the higher binding of amitriptyline to AGP when compared with nortriptyline. Interpretation of tricyclic antidepressant blood levels is clarified by obtaining assays from RBCs and plasma.

The metabolism of most tricyclic antidepressants and some phenothiazine neuroleptics is under the genetic control of hepatic cytochrome P-450IID6, which also regulates the metabolism of dextromethorphan. This study investigated the effect of treatment with amitriptyline or thioridazine on testing for genetically regulated efficiency of the metabolism of dextromethorphan and mephenytoin. One group of 33 patients was treated with 150 mg amitriptyline a day (the AMI group); 25 other patients received a daily dose of thioridazine, either 200 mg (200-THD group; n = 7) or 400 mg (400-THD group; n = 18). Before and after 10 days of this treatment, all patients were tested with 25 mg dextromethorphan and 100 mg mephenytoin to determine their pharmacogenetic status with respect to their hepatic drug oxidizing systems (cytochrome P-450IID6 and P-450 MP). Two patients were poor metabolizers (PMs) of dextromethorphan and three of mephenytoin. Treatment with either psychotropic drug was without significant effect on the metabolism of mephenytoin, but both amitriptyline and thioridazine increased significantly the metabolic ratio of dextromethorphan/dextrorphan. Thioridazine had the effect of changing the pharmacogenetic status of 15 efficient metabolizers of dextromethorphan to poor metabolizers; amitriptyline did not have such an effect. There was no significant correlation between day-11 plasma levels of thioridazine, mesoridazine, or sulforidazine and the metabolism of dextromethorphan, but there was a correlation between the metabolism of dextromethorphan and plasma levels of amitriptyline and nortriptyline. Amitriptyline (p less than 0.05), but not thioridazine, decreases the ratio of conjugated/total dextrorphan in urine.(ABSTRACT TRUNCATED AT 250 WORDS)

A 61-year-old woman, a nonsmoker, was admitted to the hospital because of endogenous depression. No concomitant disease, especially kidney or liver dysfunction, was diagnosed. After 9 days of treatment with 125 mg of amitriptyline (AMI) daily, she developed signs of a severe anticholinergic syndrome. Plasma concentrations of AMI (510 ng/ml) and nortriptyline (NOR; 320 ng/ml) were very high and the half-life of AMI was about 120 h. The debrisoquine metabolic ratio was 0.55 and 0.79 on two occasions, which shows that she had no deficiency of cytochrome P450IID6. This result was confirmed with a dextromethorphan test, analysis of restriction fragment length polymorphisms (29/29-kb fragments), and genotyping with allele-specific polymerase chain reaction (homozygous 29 kb wild-type alleles). Patients with high plasma levels of tricyclic antidepressants are usually poor metabolizers of debrisoquine. Before the administration of AMI, our patient was pretreated with fluoxetine. A slowly reversible interaction with fluoxetine or an extremely long-lasting metabolite may be responsible for the long plasma half-life of AMI.

A 34-year-old man with bipolar manic depressive illness suffered from severe adverse effects during treatment with amitriptyline, 50 mg/day. It was subsequently shown that the patient was a slow metabolizer of amitriptyline. However, he tolerated a dose of 200 mg of imipramine/day, which was necessary in order to reach a therapeutic level of about 900 nM for imipramine plus desipramine. Since both antidepressants are subject to the genetic sparteine/debrisoquine oxidation polymorphism, the patient was phenotyped with sparteine. The test performed during paroxetine treatment indicated that the patient was a poor metabolizer. Subsequent tests performed during a drug-free period, however, showed the patient to be an extensive metabolizer, with a sparteine metabolic ratio (MR) of 1.7 and 2.8 and debrisoquine MR of 2.3. It was subsequently shown that paroxetine is a potent, competitive inhibitor of 1'-hydroxybufuralol formation in a human liver microsome preparation (K1 approximately 800 nM). This patient thus illustrates two problems: (a) the erroneous phenotyping due to concurrent medication, and (b) the existence of a very slow amitriptyline elimination apparently not related to the sparteine/debrisoquine oxidation polymorphism.

The effects of valpromide on amitriptyline (AMT) and nortriptyline (NT) plasma levels were examined in 20 depressed inpatients. They all were treated with AMT, 125 mg once daily, and 10 patients also received 600 mg of valpromide daily after 10 days on AMT. In the 10 patients receiving valpromide in addition to AMT, the mean AMT level increased from 70.5 +/- 35 to 105.5 +/- 49 ng/ml (p less than 0.0003) and the mean NT level from 61.0 +/- 34 to 100.5 +/- 65 ng/ml (p less than 0.01). This increase was not related to valpromide metabolite plasma levels, nor to the age of the patients. The addition of valpromide to a stable AMT regimen may result in an increase of antidepressant plasma level with clinical implications.

A simple normal-phase (silica), high-performance liquid chromatographic (HPLC) assay of amitriptyline (AMI), doxepin (DOX), imipramine (IMI), nortriptyline (NORT), desmethyldoxepin (DESDOX), desipramine (DESIP), and protriptyline (PRO) in serum with no coelution is described here. Trimipramine and promazine were used as internal standards. Extraction of the 1.0-ml serum samples (collected in plastic) was done with Bond-Elut C18 columns. The compounds of interest were eluted with 10 mM methanolic ammonium acetate. The eluates were evaporated at 56-58 degrees C and reconstituted with 200 microliters of the mobile phase. The mobile phase was absolute ethanol-acetonitrile-tert-butylamine (98:2:0.05, vol/vol/vol). Detection of eluted drugs was at 254 nm at 0.01 absorbance units full scale (AUFS), except for PRO, which was detected at 229 nm at 0.02 AUFS. Absolute recoveries were 87-97%. A 5-micron silica (4.6 X 250 mm) HPLC column was used; results with a 10-micron silica column (3.9 X 300 mm) are also presented. Peak height ratios with trimipramine were linear for each analyte between 25 and 1200 ng/ml. Peak height ratios with promazine as the internal standard were linear for each analyte between 25 and 600 ng/ml. Detection limits under the conditions described were 2 ng/ml for AMI, DOX, and IMI, 4 ng/ml for NORT, DESDOX, and DESIP, and 10 ng/ml for PRO. Coefficients of within-day and day-to-day variation at three concentration levels were less than 9.8% and less than 11.2%, respectively. The hydroxylated metabolites of IMI, DES, NORT, and the cis isomer of DOX are discussed. Steady-state daily dosages and corresponding serum levels are presented for 69 patients. The total assay time was less than 10 min for DESIP and 12 min for PRO. This assay can be used in correlating serum levels with clinical effects, compliancy, and pharmacokinetic studies.

An improved gas-liquid chromatographic procedure for the plasma level determination of amitriptyline and nortriptyline using nitrogen-sensitive detectors is described. Derivatization of the secondary amines using trifluoroacetic anhydride greatly improves the response and reproducibility of the assay. Plasma samples containing as little as 5 ng/ml of amitriptyline and nortriptyline can be assayed precisely and reproducibly, using protriptyline as internal reference standard.

Comparison of the precision and accuracy of measurements made between December 1979 and January 1988 of tricyclic antidepressant drug concentrations in freeze-dried external quality-assurance samples of human serum showed only occasional significant differences between the different chromatographic techniques used for drug assay. Larger differences between data collected in different years correlated with changes in the source of the sample matrix and the personnel responsible for sample preparation. Measurements of amitriptyline and imipramine were more precise than those of nortriptyline and desipramine; the mean coefficients of variation of measurements were 20.2, 19.6, 26.6 and 25.3%, respectively. The data indicate the need for further interlaboratory studies directed at reducing the high level of variability in tricyclic measurements.

Homogeneous enzyme immunoassay reagents (EMIT) developed for the measurement of amitriptyline and nortriptyline in serum were modified to allow quantitation of clomipramine and desmethylclomipramine. The method was compared to a high-performance liquid chromatographic method. Between-run precision [coefficient of variation (CV)] for clomipramine in the EMIT assay for amitriptyline ranged from 2.6 to 3.2%. For desmethylclomipramine in the nortriptyline assay, the between-run CV ranged from 1.4 to 1.9%. Serum specimens from 43 patients (desmethylclomipramine) and 59 patients (clomipramine) were analyzed by both methods, with good correlation between methods. For clomipramine, recovery ranged from 100 to 102% (0-600 ng/ml range) and was 95-103% for desmethylclomipramine (0-600 ng/ml). The modified EMIT assays offered sufficient reproducibility, accuracy, and correlation with an established method for routine analysis of clomipramine and desmethylclomipramine.

A brief review of the literature surrounding the relationship between plasma levels of tricyclic antidepressants currently available in the United States and clinical efficacy is presented. While therapeutic ranges for plasma levels of some tricyclic antidepressants are reasonably well established, the relationships between plasma levels of others and clinical efficacy require further clarification. Conditions under which plasma level measurements are clinically useful and factors that alter steady-state plasma levels are discussed.

A method for the quantitative determination of the tricyclic antidepressant drug amitriptyline (AT) and its active major metabolite, nortriptyline (NT), in plasma is described. The method involves a three-step extraction procedure, no derivatization, and a rapid run on a gas--liquid chromatograph equipped with a nitrogen detector. The standard curves were linear in the range of 25-1,000 ng/ml. The lower detection limit was 2-5 ng/ml for both drugs. The method is specific for AT and NT, with a recovery of AT and NT of 68 and 71%, respectively. The precision of the method, expressed as the coefficient of variation, was 10.7% for AT and 12.9% for NT within 25--1,000 ng/ml. The method has proven to be suitable for monitoring plasma levels of AT and NT in patients, and in animals during experimental studies on pharmacokinetics after therapeutic and toxic doses of NT.

The serum concentrations of amitriptyline (AMI) and its metabolite nortriptyline (NT) were determined in 54 Saudi patients treated for long durations with a daily oral dose (0.111 to 4.167 mg/kg) of AMI. Ten of these patients were concomitantly treated with neuroleptic drugs. The mean (SEM) of the dose-normalized steady-state serum concentration (Css) of AMI in patients who received AMI alone (Group I) was [46.1 (6.5) ng X ml-1/mg X kg-1], with mean NT/AMI Css ratio of 0.962 (0.122). A significantly higher mean of Css of AMI [82.3 (18.8 ng X ml-1/mg X kg-1] was acquired for patients who concomitantly received AMI and neuroleptic drugs (Group II). However, the difference in the mean NT/AMI Css ratio between Group I and Group II [0.929 (0.197)] was not significant. Also, the results of this study indicate that Css of AMI and the NT/AMI Css ratio in Saudi patients treated for depression with AMI alone are not significantly different from those reported for subjects from Western populations.

We tested clinical samples from patients receiving four commonly monitored antidepressant drugs--amitriptyline, nortriptyline, imipramine, and desipramine--to evaluate the reliability of currently available Becton-Dickinson Vacutainer brand vacuum blood collection tubes. Serum tubes (red stopper), heparinized plasma tubes (green stopper), and two types of serum separator tubes (SST) were evaluated. Antidepressant concentrations were measured by reversed-phase high-performance liquid chromatography. Antidepressant levels obtained using fresh serum (red stopper) or plasma (green stopper) yielded equivalent results. Serum collected in red-stopper tubes was stored at 4 degrees C for 4 weeks without detectable loss of antidepressant drugs. Significant concentration decreases for amitriptyline, imipramine, and desipramine were noted when serum was collected in SST tubes to such an extent that SST tubes cannot be used for the procedure. Bias attributed to SST tubes can interfere with the usefulness of clinical results over the entire therapeutic ranges of these drugs.