Journal
JOURNAL OF SEPARATION SCIENCE
Volume 31, Issue 13, Pages 2358-2370Publisher
WILEY-V C H VERLAG GMBH
DOI: 10.1002/jssc.200800079
Keywords
benzodiazepines; biological fluids; HPLC; sequential SPE; tricyclic antidepressants
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A novel method for the simultaneous determination of six benzodiazepines (BZDs) and four tricyclic antidepressants (TCAs) in biological fluids by HPLC with UV detection at 240 nm has been developed. After a deproteinization step biological fluids were analyzed by direct injection. SPE on Nexus cartridges was also applied. Since two compounds, namely imipramine and diazepam, were coeluting, a sequential SPE protocol has been developed. BZDs were eluted by a mixture of methanol/ACN (1:1), followed by the elution of TCAs with methanol. Separation was performed on a Kromasil C-8 column (250 x 4 mm(2) id, 5 mu m) using a mobile phase of 0.05 M CH3COONH4/ACN/methanol (initial composition 55:15:30 v/v/v) at a flow rate of 1.0 mL/min delivered by a gradient program within 15 min. Colchicine was used as the internal standard (4 ng/mu L). The method was linear for all analytes up to 20 ng/mu L, with coefficients of regression between 0.996 and 0.99996. LODs and LOQs were 0.08-1.17 and 0.28-3.91 ng/mu L, respectively. Recovery was in the range of 92.8-108.7% for within-day and 91.9-109.9% for between-day assays, with RSD values lower than 10.0% for all matrices.
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