Tricyclic antidepressants, separation

F Ig u re 11.11 Chromatogram of the extraction column for tricyclic antidepressant separation in plasma. Extraction column 4 cm x4.6 mm i.d. polystyrene-divinyibcnzene, 10 pm spherical polymer. Extraction eluent 0.06 M SDS-8% (v/v) acetonitrile-0.2% triethylamine in 0.02 M phosphate buffer at pH 7.2. Recovery eluent 0.08 M SDS-56% acetonitrile-12% methanol in 0.09 M phosphate buffer at pH 3.0. Wash eluent 0.06 M SDS-90% acetonitrile. Reprinted from Ref 48 with permission of Elsevier. 

Figure 15.3 Separation of tricyclic antidepressants by using multidimensional LC-LC. Peak identification is as follows DOX, doxepin DES, desipramine NOR, noitryptylene IMI, imipramine AMI, amiti yptyline. Adapted from Journal of Chromatography, 507, J. V. Posluszny et al., Optimization of multidimensional high-performance liquid cliromatography for the deterTnination of drugs in plasma by direct injection, micellar cleanup and photodiode array detection , pp. 267 - 276, copyright 1990, with permission from Elsevier Science.

The separation was carried out on a bonded phase LC-PCN column carrying cyanopropylmethyl moieties on the surface. Thus, in contrast to the extraction process, which appears to be based on ionic interactions with the weak ion exchange material, the LC separation appears to be based on a mixture of interactions. There will be dispersive interactions of the drugs with the hydrocarbon chains of the bonded moiety and also weakly polar interactions with the cyano group. It is seen that the extraction procedures are very efficient and all the tricyclic antidepressant drugs are eluted discretely. 

Fi re 4.15 Separation of the tricyclic antidepressant amitriptyline and its major metabolites on a 10 cm x 4.6 mm I. D. column packed with Spherisorb S5H silica with methanol-aqueous ammonium acetate (9 1), pH 9.1, as mobile phase at a flow rate of about 1 ml/min. Peak identification 1 > amitriptyline-N-oxide 2 amitriptyline 3 - E-lO-hydroxyamitriptyline 4 - Z-10-hyroxyamitriptyline 5 desmethylnortrlptyline 6 nortriptyline 7 E-lO-hydroxynortriptyline and 8 - Z-lO-hydroxynortriptyline. (Reproduced with permission from ref. 271. Copyright Elsevier Scientific Publishing Co.)... 

Duverneuil and coworkers (2003) have developed a method for the determination of 11 of the most commonly prescribed non-tricyclic antidepressants and some of their metabolites these include paroxetine, fluoxetine, norfluoxetine, sertraline, citalopram, fluvoxamine mirtazapine, venlafaxine, and 0-des-methylvenlafaxine. The method involves an LLE procedure followed by an HPLC separation with photodiode-array UV detection at three different wavelengths (220, 240, and 290 nm). The total run time was 18 min. The extraction recoveries were calculated to be in the range of 74-109% and the lower limit of detection (LLOD) reported was 2.5-5 ng/ml. A method published by Tournel and associates (2001) also reported the simultaneous determination of several newer antidepressants by RP-HPLC with UV detection. The compounds were isolated from human serum using an LLE process. The LLOQ ranged from 15-50 ng/ml depending on the analyte of interest. The total run time for all compounds eluted was approximately 20 min. 

Tricyclic antidepressants act both presynaptically and postsynaptically on two major systems, the norader-nergic and the serotonergic. The actions of TCAs in each system will be discussed separately. Figure 23.2 shows an overall schematic of the receptor blockade of TCAs. 

Tricyclic antidepressants, major tranquillizers used in mental hospitals, are separated at UV, 254 nm, on Ci8 using 55% An/water at pH 5.5 with pentane sulfonate. Since these are very basic compounds, it is necessary to use hybrid or heavily end-capped columns and their separation benefits from organic modifiers, such as nonyl amine. 

K. Salomon, D. S. Burg, and J. C. Helmer, Separation of seven tricyclic antidepressants using capillary elecrophoresis, J. Chromatogr., 549 375 (1991). 

An example of the use of PDA UV libraries to help in the identification of peaks is provided by the identification and quantification of tricyclic antidepressants.7 Although the structures of the compounds are very similar, making separation fairly difficult, the UV spectra of most tricyclic antidepressants are fairly distinct, as shown in Figure 7.4. On the occasions where the UV spectra are similar, the retention times are sufficiently different to prevent misidentification. 

An interesting (if not fully understood) phenomenon has been observed by Smith et al. as strong cation-exchange (SCX) phases were used to separate tricyclic antidepressants [47], A certain focusing effect, which is not reproducible, produced peaks of staggering efficiencies, millions of plates per meter, the... 

Enlund et al. [130] have designed and evaluated polyacrylamide-based monolithic columns. On the best stationary phase, which has a molar ratio of 1 80 between the two functional ligands, vinylsulfonic acid and isopropylacrylamide, efficiencies of up to 200,000 plates/m were obtained for the separation of three tricyclic antidepressants. 

Figure 30 MIP-CEC separation of a simulated combinatorial library consisting of several tricyclic antidepressants. Conditions capillary i.d. 100 mm Lm 33 cm Z.bcd 22.5 cm eluent acetonitrile/10 mM Na acetate pH 3.0 (98/2) with 0.02% trifluor-acetic acid and 0.015% triethylamine (v/v) voltage+ 30 kV constant injection + 2 kV, 2 s column temperature 50°C. (Reprinted from Ref. 131, with permission.)...

Mass spectrometric detection has also been directly interfaced with microchip separations for drug detection. These studies, detecting imipramine and desipramine in fortified human plasma, show analysis of spiked analytes in clinical sample matrices for drug detection [3]. These widely used tricyclic antidepressants inhibit the reuptake of the neurotransmitters serotonin and norepinephrine in the central nervous system. Unfortunately, the 5-mg/mL detection limit found for these antidepressants with this method is not low enough to detect typical clinical levels of the drugs. Combinatorial library characterization and preclinical drug delivery studies should benefit, however, since the concentra-... 

Separations involving cis/trans isomers can also be accomplished by employing NPC. An example of this application is the separation of tricyclic antidepressant doxepin, which is marketed as a mixture of geometric isomers in a cis/trans ratio of 15 85 [41], When a spherisorb silica column is used with a hexane-methanol-nonylamine mobile-phase system, the cis isomer of doxepin elutes first. The structures of the two isomers and the chromatographic separation are shown in Figure 5-7. NPC has also been successfully employed in the separation of cis/trans isomers of steroids. Four diastereomers... 

Figure 18-5. The separation of therapeutic tricyclic antidepressants on PBD-coated zirconia at different temperatures. Solutes 1, lidnocaine 2, quinidine 3, norephedrine 4, tryptamine 5, amitriptyline 6, nortriptyline. (Reproduced from reference 52, with permission. Copyright 1997, American Chemical Society.)...

Particle-loaded membranes with embedded Cg hydrophobic adsorbents were intensively investigated for several dmg separations. Tricyclic antidepressants, antiarrhythmic dmgs, amiodarone and its metabolite desethylamiodarone, and mexiletine and flecainide were extracted from serum using a 11 mm Cg membrane adsorber with recoveries ranging from 82% to 98% [14,220]. 

Figure 6-2 Chromatogram from an HPLC reversed-phase separation of tricyclic antidepressants with the use of a UV photometer detector set at 2l5nm. Signal is displayed at 0.1 AUFS, HPLC high-performance liquid chromatography UV, ultraviolet AUFS, absorbance units full scale. (Courtesy Vydac/The Separations Group, Hesperia, Calif.)...

Analysis by gas chromatography-mass spectrometry (GC/MS) using selected ion monitoring is the reference method, Methods for trazodone and amoxapine are also available. HPLC has been successfully used for analysis of tricyclic antidepressants, although separation and selectivity are problems with this technique. ... 

As it has a lower polarity, this phase often gives separations in a similar way to silica, but the k values are smaller for the same mobile phase.It can be used as a reversed phase as well in this mode it is less retentive than Cg or Cig phases. It is particularly selective towards components with double bonds and towards tricyclic antidepressants. An application from this latter field is presented in Figure 11.2. 

Figure 11.2 Separation of tricyclic antidepressants on a nitrile phase [reproduced with permission from G.L. Lensmeyer, D.A. Wiebe and B.A. Darcey, J. Chromatogr. Sci., 29, 444 (1991)]. Conditions sample, serum extract from a patient who received clomipramine column, 25cm x 4.6mm i.d. stationary phase, Zorbax Cyanopropyl, 5-6pm mobile phase, 1.2ml min water-acetonitrile-acetic acid-n-butylamine (600 400 2.5 1.5) temperature, 45°C UV detector, 254nm. Peaks M = metabolites of clomipramine 1 =trimipramine (internal standard) 2 = des-methylclomipramine 3 = clomipramine.

In drug discovery, we often avoid elevated temperatures because polar organic molecules tend to be thermally labile. Over the modest ranges used, temperature usually has little effect on retention. However, it has been observed that subtle changes in temperature can dramatically affect selectivity or relative retention. It is not uncommon for closely related compounds to show very different responses to 5-10°C changes in temperature. At 35°C, three tricyclic antidepressants coeluted, but at 40 °C, they were baseline separated (33). 

Piperaki, S. Parissi-Poulou, M. Koupparis, M. A separation study of tricyclic antidepressant drugs by HPLC with p-cyclodextrin bonded stationary phase. J.Liq.Chromatogr, 1993, 16, 3487-3508 [simultaneous chloripramine, doxepin, imipramine, maprotiline, nortriptyline, protriptyline]... 

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