Selection of Mobile Phase in High-Performance Liquid Chromatographic Determination for Tricyclic Antidepressants in Serum

Shin, Myung Geun; Kim, Soo Hyun; Shin, Jong Hee; Suh, Soon Pal; Ryang, Dong Wook

Abstract

BACKGROUND
Optimal use of tricyclic antidepressants (TCAs) requires serum monitoring to determine if the appropriate therapeutic range has been attained and to assess possible side effects. This study was to evaluate the resolution capacity of the following four mobile phases which were previously reported; mobile phase I (methanol, acetonitrile and 5 mmol/L Na2HPO4: 41/15/44 by volume), II (methanol, acetonitrile and 5 mmol/L Na2HPO4,: 15/60/25 by volume), III (acetonitrile and 2-propanol: 95/5 by volume) and IV (methanol and n-butylamine: 99.5/0.5 by volume).
METHODS
Amitriptyline (AT), nortriptyline (NT), imipramine (IMI) and doxepin (DOX) were used for the selection of appropriate mobile phase in high performance liquid chromatographic (HPLC) determination. TCAs were extracted from serum with hexane, isoamyl alcohol (99:1). The drug was re-extracted into 0.1 N HCl and an aliquot was injected into the HPLC. The analytical column was C-18 reversed phase column (3.9 mm x 30 cm; Waters, USA) with the flow rate of 1.5 mL/min. The UV detector signal was monitored at 254 nm.
RESULTS
Mobile phase I disclosed 9.8-15.8 retention time (min), 5.1-8.8 capacity ratio and 1.0-2.2 resolution factors for the above four TCAs. Precision studies using this mobile phase demonstrated a coefficient variation of 2.4-4.7% in the concentration range of 500-125 ng/mL. Analytical recovery of AT and IMI was 85-90% at a concentration of 125 ng/mL and 250 ng/mL.
CONCLUSIONS
Mobile phase I provided a reliable and excellent resolution of TCAs in the use of HPLC with the C-18 reversed phase column and UV absorbance detector.