Laboratory Analysis Of Tcas

It is important to stress the need to avoid serum or plasma separator tubes when collecting samples for analysis of TCAs. Nyberg and Martensson (18) studied several types of blood collection tubes for stability of amitriptyline, imipramine, clomipramine, and their mono-demethylated metabolites collected in these tubes. Ethylenediaminete-traacetic acid (EDTA) tubes were most suitable and serum separator gel tubes were unsuitable because of loss of more than 40% of drug concentrations on storage. The losses were not caused by redistribution between blood cells and plasma but occurred mainly because of sample contact with serum or plasma separator gel or the caps of the tubes. Dasgupta et al. (19) studied Greiner serum separator gel tubes for stability of TCAs and many other drugs, and concluded that these tubes are not suitable for blood collection for analysis of TCAs.

Qualitative, semiquantitative, and quantitative methods are available for the analyses of TCAs. There are no reliable spot tests available for TCAs. Common methods of analysis for TCAs include immunoassays, thin layer chromatography (TLC), high-performance liquid chromatography (HPLC), and gas chromatography (GC). Immunoassays provide a rapid method for determination of TCAs. Owing to their faster turnaround time and unavailability of specific methods on an emergency basis, immunoassays provide rapid clinically useful information, particularly in overdose situations. Two commonly used formats are individual assays for a particular TCA and "total TCAs." The individual immunoassays employ monoclonal antibodies whereas "total TCAs" assays utilize polyclonal antibodies. The problem with these assays is the considerable cross-reactivity of the tertiary and secondary amines, for example, imipramine and amitriptyline cross-react with each other in their individual assays. Similarly, at therapeutic concentration, desipramine cross-reacts with nortriptyline assay and vice versa. Many structurally related drugs, including clomipramine, cyclobenzaprine, doxepin, and chlorpromazine also cross-react with these assays. Although these assays may provide reliable results in patients treated with monotherapy, these assays are not suitable in patients receiving any drug, which may cross-react with the assays. Owing to common interferences in immunoassays, both positive and negative data should be interpreted according to manufacturer guidelines and in context to clinical information.

TLC can reliably detect TCAs. One of the common TLC systems is Toxi-Lab (Varian Inc., Palo Alto, CA). Gas chromatographic methods coupled with flame ionization detector (FID), nitrogen phosphorus detector (NPD), or mass spectrometer (MS) are widely used for screening and quantification of TCAs. Owing to their lipophilic characters, TCAs are good candidates for liquid-liquid extraction, although numbers of solid-phase extraction procedures have also been reported. Columns are typically fused silica capillary columns with bonded non-polar to intermediate polarity methyl silicone liquid phases (0-50% phenyl). Although sample derivati-zation is not necessary, it improves chromatographic separation. Trifluoroacetyl and heptafluorobutyryl are commonly used derivatives. However, these derivatives are not very stable. Way et al. (20), using stable isotope dilution GC-MS, found that 4-carbethoxyhexafluorobutyryl chloride derivatives are more stable than trifluoroacetyl derivatives.

HPLC is another widely used method for the analysis of TCAs. Problems posed in GC by polar secondary amines and hydroxy metabolites are easily overcome by HPLC. As reported on CAP proficiency-testing surveys, HPLC with UV detection is the most common method for quantitative analysis of TCAs. The columns predominantly used in HPLC are C18, C8, phenyl, and CN, and permit simultaneous determination of tertiary and secondary amines. Common mobile phases are phosphate buffers with or without ion-pairing agents. HPLC methods with normal phase silica and aqueous base mobile phase with fluorescence or electrochemical detection methods are also available (21). Occasionally, HPLC will not separate all the drugs of interest. Application of HPLC-mass spectrometry in TCA analysis is relatively new and can overcome such problems. A sensitive and specific HPLC-MS-MS method has been described for the rapid identification and quantitation of seven TCAs: amitriptyline, nortriptyline, doxepin, dosulepin, dibenzepin, opipramol, and melitracen. The method uses direct injection and on-line removal of proteins and other large biomolecules with total analysis time of 12min (22).

3.1. Interferences in TCA Assays

Rapid toxicological screening by immunoassays is a common practice in the clinical laboratories. False-positive results because of cross-reacting compounds in drug assays may lead to misdiagnosis and mismanagement of a patient. The issue becomes even more serious when such false-positive result is found in a child and child neglect or child abuse is suspected. Most of these false-positive results are due to cross-reactivity in TCA immunoassays with structurally similar drugs. The drugs which are shown to interfere with TCA immunoassays are shown in Fig. 1.

Carbamazepine, structurally close to TCAs, is known to interfere in various TCA immunoassays. Several cases of false-positive TCAs because of carbamazepine have been reported (23-26). Fleischman et al. (23) report a case of a 16-year-old girl who was found unresponsive and had a remote history of seizures. The patient's serum screen for TCAs was positive. ECG showed no evidence of QRS or axis deviation. The validity of the TCA value was questioned and carbamazepine level was measured and

Amitriptyline

Amitriptyline

Cyproheptadine ch3

Cyproheptadine

Cyclobenzaprine

Cyclobenzaprine

Thioridazine
Chlorpromazine
Diphenhydramine

Fig. 1. Chemical structure of amitriptyline and some drugs that interference with tricyclic antidepressants immunoassays.

the value was found to be 17 ^g/mL. Further investigation showed that the positive TCA screening result was due to carbamazepine in the specimen. Chattergoon et al. reported two patients with history of ingestion of carbamazepine who tested positive for TCAs using the fluorescence polarization immunoassay (FPIA) screening assay. The apparent TCA concentrations were 80 and 130ng/mL, respectively (25). The HPLC analysis failed to detect any TCAs in the serum. Tomaszewski et al. (26) reported three

Garg false-positive cases of TCAs, on the Triage Panel Immunoassay System, because of interference from iminostilbene (a carbamazepine metabolite).

Quetiapine, an antipsychotic drug, has been found to interfere in TCA immunoassays. Sloan et al. (27) reported a case where a 34-year-old patient tested positive for TCAs by immunoassay. The patient was not prescribed any TCAs, and he denied their use. The patient was receiving quetiapine. The possibility of quetiapine interference in TCA assay was raised because it is structurally similar to TCAs. Various concentrations ranging from 1 to 10 ^g/mL were tested for interference in Diagnostic Reagent Inc. (Fremont, CA) (now called Microgenics) immunoassay on the Hitachi 911 analyzer. Cross-reactivity was found to be 4.3%. The authors also tested olanzapine, another antipsy-chotic, and did not find any significant cross-reactivity with the assay. Hendrickson and Morocco (28) investigated three common TCA immunoassays (Microgenics, Syva Rapid Test, and Biosite Triage) using quetiapine solution. They also used urine samples from a patient overdosed with quetiapine as well as a patient with therapeutic concentration of the drug. Syva and Microgenics immunoassays, but not Triage immunoassay, tested positive in both the overdose and the therapeutic samples. Syva and Microgenics immunoassays were positive at urine levels of 100 and 10 ^g/mL, respectively, whereas the Triage immunoassay was negative in solutions up to 1000 ^g/mL. The study concluded that quetiapine might cause false-positive results in certain TCA immunoassays in both therapeutic and overdose situations and significant variations exist between different immunoassays so far quetiapine cross-reactivity is concerned. Schussler et al. (29) showed false-positive results by quetiapine in Abbott Laboratories' fluorescence polarization immunoassay on TDx/TDxFlx. Caravati investigated Abbott's FPIA, and Syva as well as STAD-ACA qualitative TCA immunoassays for cross-reactivity with quetiapine using spiked plasma samples, and found all these assays cross-react with quetiapine in a concentration-dependent manner (30).

Several reports of interference of phenothiazepine in TCA immunoassays have been published. Ryder and Glick (31) reported a case where a patient who ingested thioridazine and flurazepam tested positive for TCAs by immunoassays. Investigation showed that false-positive TCA result was due to thioridazine. It is important to note that even therapeutic concentration of thioridazine (125 ng/mL) produces a false-positive serum TCA screen result. Maynard and Soni (32) reported false elevations of imipramine and desipramine in HPLC (cyanopropyl column) caused by thiori-dazine. High therapeutic (200-300 ng/mL) or toxic concentrations of chlorpromazine produce false-positive result in the enzyme-multiplied immunoassay technique (EMIT). In another study, false-positive results were obtained with high concentrations of thior-idazine (4000 nM), chlorpromazine (300 nM), and trimeprazine (5000 nM) (33).

Sorisky and Watson (34) reported a case where a 21-year-old female who ingested 2 g of diphenhydramine tested positive for TCAs using EMIT. Unlike certain phenoth-iazines that have tricyclic structure, diphenhydramine is an ethanolamine.

Wians et al. reported a case of a 14-year-old girl who ingested approximately 120 mg of cyproheptadine, an antihistamine and serotonin antagonist with anticholinergic and sedative properties. The patient tested positive for TCAs by EMIT (35). In vitro studies indicated that a cyproheptadine concentration of 400 ^g/L may cause false-positive TCA results. However, serum obtained from a volunteer who was given a 12-mg dose of cyproheptadine for 3 days tested negative for TCAs. Yuan et al. (36) report a pediatric case of false-positive TCAs because of cyproheptadine and found that the false-positive test was mainly because of cyproheptadine metabolite. Cyproheptadine has tricyclic structure very similar to tricyclic structure of TCAs (Fig. 1).

Cyclobenzaprine and its major metabolite nor-cyclobenzaprine differ from amitriptyline and nortriptyline only by the presence of a double bond in the cyclo-heptane ring and are known to interfere with immunoassays and HPLC. Wong et al. (37) reported positive interferences by cyclobenzaprine in both the Syva EMIT assay and the HPLC. In an HPLC, cyclobenzaprine co-eluted with amitriptyline and nor-cyclobenzaprine eluted very close to nortriptyline. These interferences could be overcome by GC-MS after derivatization with trifluoroacetic anhydride, as these compounds had distinguishable mass spectra. A review on cyclobenzaprine interference in TCA assay has been published (38).

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