Tips in dealing with interferences in tcas

Over time, several methods have been developed to eliminate or reduce the inferences in TCA assays. Dasgupta et al. (39) proposed a mathematical model for the estimation of TCA concentration in the presence of carbamazepine using the FPIA. Using sera from 30 patients who were receiving carbamazepine but no TCAs, and negative sera spiked with carbamazepine and its metabolite, they determined apparent TCA concentrations. In sera of patients, the carbamazepine concentrations ranged from 1.4 to 20.9 ^g/mL and apparent TCAs concentrations ranged from 31.8 to 130.1 ng/mL. From the known carbamazepine concentrations and apparent TCA concentrations, they developed mathematical equation for estimation of TCAs in the presence of carbamazepine. They tested the equation by spiking carbamazepine-containing patients' samples with known concentrations of TCAs. There was a good agreement between the calculated and the targeted TCA concentrations. The differences between the predicted and observed values were <10%. This mathematical modeling was feasible because TCAs, even at very high concentrations, do not show interference with the carbamazepine FPIA. The carbamazepine showed significantly higher interference than its metabolite carbamazepine 10, 11-epoxide. Therefore, the authors cautioned the use of the equation in patients who may accumulate higher concentration of carbamazepine 10, 11-epoxide, e.g., renal patients. Also, the equation may not be valid at high concentrations of carbamazepine. The highest carbamazepine concentration from a patient sample was 20.9 ^g/mL. When a carbamazepine concentration of 40 ^g/mL was tested, the difference between observed and expected concentration was 18%.

Adamczyk et al. (40) described a method for removal of phenothiazine interference in TCA assays. The method involved alkalinization of phenothiazine containing serum sample followed by treatment with isoamyl alcohol to dissociate the analyte from serum proteins. The analyte was extracted with decane and transferred to an acidic buffer (0.1 M Gly-Gly, pH 3) containing chloramines-T. This results in selective oxidation of the phenothiazine sulfur atom in an acidic buffer system. The aqueous layer was analyzed for TCA by using the FPIA and TDx analyzer. This method allowed accurate quantification of the TCAs in the presence of 1000 ng/mL chlorpromazine or desmethyl chlorpromazine.

Cyclobenzaprine, a commonly used skeletal muscle relaxant, interferes with immunoassays and may co-elute with amitriptyline in HPLC. However, amitriptyline and cyclobenzaprine can be distinguished using HPLC with diode array detector, as these drugs have different UV spectra. Puopolo and Flood (41) used dual wavelength (214 and 254 nm) spectrometry for detection of cyclobenzaprine interference in TCA HPLC.

In TLC, cyclobenzaprine may co-migrate with amitriptyline but can be distinguished by difference in fluorescence at stage III. Amitriptyline gives pink fluorescence, whereas cyclobenzaprine has orange fluorescence. Sometimes it is hard to distinguish between pink and orange color on TLC. Looking for amitriptyline and cyclobenzaprine metabolites is very helpful in distinguishing the presence of these drugs on TLC.

On capillary GC, cyclobenzaprine and amitriptyline are generally well separated. However, in an overdose situation, when peak size is too large and peak shape is not symmetrical or column performance is not optimal, the two drugs can co-elute and retention times may shift, causing confusion. In that case, diluting and reanalyzing the sample generally resolves the issue. Upon GC-MS, cyclobenzaprine and amitriptyline show certain similarity in mass spectra (Fig. 2). In both drugs, m/z 202 and 215 ions are prominent, and these ions ratios can be used to differentiate these drugs.

Fig. 2. Electron impact ionization mass spectra of amitriptyline and cyclobenzaprine. They share number of common ions and cause confusion in identification, particularly if there is baseline noise. Careful examination of ions (e.g., ratio of 202/215) may help distinguishing the spectra. To show other ions, abundance of ion 58 has been truncated.

Fig. 2. Electron impact ionization mass spectra of amitriptyline and cyclobenzaprine. They share number of common ions and cause confusion in identification, particularly if there is baseline noise. Careful examination of ions (e.g., ratio of 202/215) may help distinguishing the spectra. To show other ions, abundance of ion 58 has been truncated.

A method to eliminate adsorption loss of TCAs during solvent extraction and evaporation has been described. The authors reported that the loss can be as high as 50%, and addition of as little as 0.05% diethylamine to the extract before evaporation completely eliminates the adsorption loss of amitriptyline, nortriptyline, imipramine-desipramine, doxepin, and nordoxepin (42).

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