Has anyone tried extracting lofepramine from serum ? Given it's structural similarity to imipramine, it should have extracted easily using our existing liquid - liquid TCA method (as imipramine does), but it's very poor in comparison. I've also tried SPE using a mixed mode cartridge, again the other TCAs extract but not lofepramine. Any ideas would be welcome.

The LLoQ for the following was 2.5 ng/ml. I hope it is of some help.

A 100 µl aliquot of sodium borohydride solution was added to 1 ml of plasma standard or sample in a disposable plastic tube and vortexed to mix. The tubes were incubated at room temperature for 15 minutes. The reaction was stopped by the addition of 1 ml of internal standard solution (93 ng/ml imipramine in 0.1 M HCl).

The extraction procedure was carried out automatically using a Gilson ASPEC. Varian Isolute columns (1 ml capacity, containing 100 mg cyanopropyl sorbent, end capped) were conditioned with 1 ml of methanol followed by 1 ml of 0.1 M HCl. The whole derivatized samples (2.1 ml) were applied to the columns and blown through under pressure. The columns were washed with 1 ml of water and the amino alcohol derivative of lofepramine and the internal standard were eluted with a mixture of methanol in HPLC mobile phase (30/70 v/v). 20 ìl of this solution was injected onto the HPLC column for chromatography.

Equipment and Operating Conditions

Mobile phase: Acetonitrile : methanol : 0.02M hydrogen phosphate buffer (55 : 5 : 40) - the pH was adjusted to 7.3 with sodium hydroxide
Flow rate: 1.0 ml/min
Temperature: 59C
Injector: Gilson ASPEC
Run time: 11 min (including extraction time)
Volume: 20 µl
Analytical Column: Spherisorb Cyano 5 µm (150 x 4.6 mm)
Detector: Coulochem 5100A with a 5011 Analytical cell
Output: 10 mvfsd
Ranges: Electrode 1 0.50 V
Electrode 2 0.80 V
Gain: Electrode 2 50 x 100 µA
Filter: 10 seconds
Auto zero: -15%