Isotope Dilution Gas Chromatographic-Mass Spectrometric Measurement of Tricyclic Antidepressant Drugs. Utility of the 4-Carbethoxyhexafluorobutyryl Derivatives of Secondary Amines

Journalof AnalyticalToxicology,Vol. 22, September1998 Isotope Dilution Gas ChromatographicMass SpectrometricMeasurementof Tricyclic AntidepressantDrugs. Utility of the 4-CarbethoxyhexafluorobutyrylDerivativesof SecondaryAmines Barbara A. Way 2, Douglas Stickle1,2, Mary E. Mitchell 4, John W. Koenig4, and John Turk1,2,3,* IMass SpectrometryResource,Departmentsof 2pathologyand 3Medicine, WashingtonUniversity School of Medicine, St. Louis, Missoufl 63110 and 4Barnes-JewishHospital, St. Louis, Missoufl 63110 [Abstract Stable isotope dilution gas chromatographic-mass spectrometric (GC-MS) measurement of tricyclic antidepressants (TCA) is a useful alternative to high-performance liquid chromatography (HPLC) methods when interfering substances prevent accurate quantitation by HPLC. For satisfactory GC-MS analysis, secondary amine TCA must be derivatized. Commonly employed trifluoroacetyl and heptafluorobutyryl derivatives are relatively unstable and cause rapid deterioration of capillary GC columns. Therefore we examined 4-carbethoxyhexafluorobutyryl chloride (CHFB-CI) as an alternative derivatizing agent and developed a stable isotope dilution GC-MS method employing ring-labeled [2H4]-desipramine and [ZH4]-imipramine internal standards, which permits measurement of desipramine, nortriptyline, imipramine, and amitriptyline in plasma samples containing one or all of these analytes. The GC-MS assay is linear for each analyte from the lower limit of quantitation (25 ng/mL) up to 1500 ng/mL and correlates well with HPLC measurements. The GC-MS analytic coefficient of variation was 9.7 _+1.3% for all analytes considered together. Although interferences are observed in the HPLC assay, thioridazine, perphenazine, cyclobenzaprine, and norcyclobenzaprine do not interfere with GC-MS measurements of the TCA examined here. The stability of the CHFB derivative of secondary amine TCA was found to be superior to that of the trifluoroacetyl derivatives of these compounds. Introduction Tricyclic antidepressants (TCA)are psychoactive drugs that are widely used to treat depression (1). Antidepressant efficacymay be optimized if plasma TCA concentrations are maintained within a target concentration range (1,2). The most important adverse side 9 Addressfor correspondence: JohnTurk, Washington University School of Medicine, Box 8127, 660 S. Euclid Ave., St. Louis, MO 63110. 374 effects of tricyclic antidepressants involve the central nervous and cardiovascular systems (3-5). Central nervous system toxicity, including delirium and convulsions, and cardiovascular effects, including conduction delays and arrhythmias, may occur more frequently at plasma TCA concentrations onsiderably above the target concentration range (1,4). Laboratory measurement of the plasma TCA concentrations is therefore commonly used for therapeutic monitoring and may provide adjunctive information for assessment of the potential severity of acute overdosage. Several analytic methods have been developed for measuring plasma levels of TCA. These methods include thin-layer chromatography with densitometry (6), radioimmunoassay (7), highperformance liquid chromatography (HPLC) with UV detection (8), and gas chromatography-mass spectrometry (GC-MS) (8-13). GC-MS can provide accurate quantitation of TCA when other substances such as cyclobenzaprine (14) or phenothiazines interfere with alternate measurement methods. When tertiary amine TCA (e.g., amitriptyline and imipramine) are administered, it is customary to measure both parent drugs and desmethyl metabolites because such metabolites are pharmacologically active (1). The desmethyl metabolites are secondary amines and require derivatization for satisfactory GC or GC-MS analysis. Several methods for the derivatization of secondary amine TCAfor GC-MS analysis have been described (8-13). Derivatization reagents used for this purpose include heptafluorobutyric anhydride (9,11), and trifluoroacetic anhydride (TFAA) (8,10,12,13). The resultant derivatives are somewhat moisture-sensitive, and residual traces of derivatizing reagent can adversely affect capillary GC column lifetime. The reagent 4-carbethoxyhexafluorobutyryl chloride (CHFB-CI) has been used to form stable derivatives of drugs with secondary amine groups, such as methamphetamine (15), and the stability of the resultant derivatives permits removal of excess derivatizing agent by the addition of protic solvents. The CHFB derivatives are also less volatile than other commonly employed derivatives and Reproduction (photocopying) of editorial content of this journal is prohibited without publisher's permission. Journal of Analytical Toxicology, Vol. 22, September 1998 tend to yield mass spectra with more abundant ions at high massto-charge ratios.These propertiesfacilitateeliminationof interference from extraneouslow molecularweight contaminants. We examined the suitability of CHFB derivativesof TCAwith secondary amine groups for isotope dilution GC-MS measurements and have developedan assay to measure desipramine and nortriptyline and the tertiary amine TCA imipramine and amitriptyline in plasma samples. Ring-labeled[2H4]-desipramine and [~H4]-imipraminewere used as the internal standards. Materials and Methods Reagents Desipramine(10,11-dihydro-N-methyl-5H-dibenz[b,f]azepine-5propanarnine), nortriptyline (3-(10,11-dihydro-5H-dibenzo[a,d]cyclohepten-5-ylidene)-N-methyl-l-propanamine),imipramine (10,11-dihydro-N,N-dimethyl-5H-dibenz[b,f]azepine-5propanamine), amitriptyline (3-(10,11-dihydro-5H-dibenzo[a,d]cyclohepten-5-ylidene)-N,N-dimethyl-l-propanamine),and trimipramine (5-(3-dimethylamino-2-methylpropyl)-10,11-dihydro-5Hdibenz[b,f]azepine)were obtained from Alltech-AppliedScience (Deerfield, IL).The deuterium-labeledcompounds (2,4,6,8)-2H4imipramineand (2,4,6,8)-2H4-desipramine,each labeledon the ring structure, were obtained from Cambridge Isotope Laboratories (Andover,MA)for use as internal standards. The derivatizingagent 4-carbethoxyhexafluorobutyrylchloride (CHFB-C1)was obtained from PCR (Gainesville,FL). Toluene,acetonitrile, and methanol were from Burdick& Jackson (Muskegee,MI).Isoamylalcoholand n-hexane were from Mallinckrodt-Baker,Inc. (Paris, KY).Ethyl acetate, mono- and dibasicpotassium phosphatewere from Fisher Scientific (Pittsburgh, PA).Sodium tetraborate decahydratewas fromAldrichChemical(Milwaukee,WI).Controlmedia(Lyphochek Benzo~CA Controls I and 2) were from BioRad (Anaheim,CA). These media contain desipramine, nortriptyline,imipramine,and amitriptyline at defined concentrations. A calibrator solution (TricyclicSerum Control) was obtained from Quality Assurance ServiceCorp. (Augusta,CA).This solution contains desipramine, nortfiptyline, imipramine, and amitriptyline (250 ng/mL each). Thioridazine,perphenazine,cyclobenzaprine,norcyclobenzaprine, trifluoroacetic anhydride (TFAA),sodium hydroxide, and fetal bovineserum werefrom SigmaChemicalCo. (St. Louis,MO). Preparationof samplesfor GC-MS For (...truncated)