Determination of the tricyclic antidepressants amitriptyline, nortriptyline, imipramine, desipramine, clomipramine and desmethylclomipramine in dried blood spots using LC-MS/MS

Background: Therapeutic Drug Monitoring (TDM) of Tricyclic Antidepressants (TADs) is considered useful in patients with major depressive disorders, since these drugs display large individual differences in clearance and therapeutic windows of these drugs are relatively small. We developed an assay for determination of amitriptyline (AMI), nortriptyline (NOR), imipramine (IMI) desipramine (DESI), clomipramine (CLOM) and desmethylclomipramine (DES-CLOM) in dried blood spots (DBS). Methods: A fast and robust LC-MS/MS method was developed and fully validated for simultaneous determination of AMI/NOR, IMI/DESI, or CLOM/ DES-CLOM in DBS. Nine millimetre circles were punched out from DBS collected on Whatman DMK-C paper and mixed with acetonitril:methanol 1:3 containing the internal standard. The extract was analyzed by LC-MS/MS Results: The assay was found to be linear in the range 20-500 mcg/L for all compounds. The Inter-assay variability (VC) and bias values are displayed below: The influence of punch position and blood volume per spot did not influence the results, but a low hematocrit (10% negative bias for all compounds. Comparison of venous blood samples with DBS samples revealed linear correlations and DBS/plasma ratio's ranging from 1.15 to 1.25. Conclusions: This fully validated method is suitable for determination of TADs in DBS and applicable for TDM. The method will be used to determine NOR in the Dutch CYSCE multicenter trial (ClincialTrail.gov Identifier NCT01778907) in which the effect of CYP2D6 genotyping combined with TDM on time to reach adequate blood drug levels will be investigated in depressed elderly patients.