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Amiodarone and Desethylamiodarone Bind to Barrier Gels in Serum Separator Blood Collection Tubes

Serum separator blood collection tubes are widely used because they offer numerous advantages. Barrier gel facilitates separation of serum from blood cells and allows samples to be taken directly from the primary collection tube. The collection tube can also be stored for reanalysis or further testing. As previously documented (1), many analytes are stable in serum tubes containing barrier gels. However, serum concentrations of several drugs, e.g. phenytoin, lidocaine and quinidine, have been reported to decrease significantly when the serum is stored in serum separator tubes (2–6). During routine testing, we had observed that concentrations of amiodarone and desethylamiodarone were consistently low or undetectable in serum samples that had been stored in serum separator tubes. We therefore decided to investigate the stability of amiodarone and desethylamiodarone in such tubes. Pooled human serum samples were spiked with amiodarone and desethylamiodarone (2.5 mg/l each) or with phenytoin (15 mg/l). Aliquots (5 ml each) were transferred either into tubes with separator gel from two different manufacturers (SSTTM, Becton Dickinson, Heidelberg, Germany, or S-Monovette Gel, Sarstedt, Nürnbrecht, Germany) or into serum tubes without gel (S-Monovette without additives, Sarstedt). Samples were gently agitated on a shaker at room temperature for up to 72 h. In each of three similar experiments, 400 μl aliquots of each sample were analysed after 0 h, 24 h, 48 h and 72 h of storage. Amiodarone and desethylamiodarone were determined by reversed-phase high performance liquid chromatography (HPLC) according to Berger et al. (7). Phenytoin concentration was measured by fluorescence polarisation immunoassay (FPIA, AXSYM, Abbott, Wiesbaden, Germany). Between-run imprecision for the determination of amiodarone was 3.8% at an analyte concentration of 2.5 mg/l and 3.4% at a concentration of 1 mg/l. For desethylamiodarone, between-run imprecision was 4.5% at 2.4 mg/l and 3.8% at 1 mg/l. For phenytoin, betweenrun imprecision was 2.9% at 15.5 mg/l and 3.4% at 5.1 mg/l. For comparison, blood samples from a patient on continuous amiodarone therapy were drawn into tubes containing EDTA (Sarstedt) and stored at room temperature. Some of the samples were stored as whole blood in the original tube. The rest of the samples were centrifuged and the resulting plasma transferred into a second tube for storage. Aliquots from both whole blood and plasma were analysed after 0 h, 24 h, 48 h and 72 h of storage. The concentrations of amiodarone and desethylamiodarone declined markedly when samples were stored in tubes of two different manufacturers both containing serum separator gel. The decrease in amiodarone and desethylamiodarone concentration in the presence of gel was more pronounced than the decrease in phenytoin. In contrast, the serum concentrations of all three analytes were nearly constant in the absence of gel (Table 1). In a separate experiment, concentrations of amiodarone and desethylamiodarone declined only slightly over 72 h when samples were stored as either whole blood or EDTA-plasma (Table 2). The observed reductions of amiodarone and desethylamiodarone occur because the serum separator gel absorbs these analytes. The drugs bound to the separator gel could be re-extracted with methanol and detected by HPLC (data not shown). Siebers et al. (8) did not observe the binding of amiodarone and desethylamiodarone to serum separator gel. However, their study differs from the current study with respect to sample storage time. In the Siebers study, the blood sample tubes sat for only 30 min at room temperature before being centrifuged; after centrifugation they were held at –20°C until analysis. In contrast, our samples sat at room temperature for up to 72 h before they were analysed. After only 24 h storage in tubes containing barrier gel, the samples showed a marked decrease in the concentration of amiodarone and desethylamiodarone. These data suggest that blood collection tubes with serum separator gel should not be used for therapeutic drug monitoring of amiodarone. Instead of serum, we recommend using plasma samples because the analyte is most stable in plasma. Tubes containing a new gel (SSTTM II) have recently been introduced. The suitability of these tubes for analysis of amiodarone is yet to be investigated.