LC/MS/MS Method for the Determination of Tricyclic and Tetracyclic Antidepressants in Human Urine

摘要

Due to the extensive conjugation of tricyclic antidepressants it was necessary to develop and validate a method with an efficient hydrolysis step. The IMCSzyme was selected due to its increased efficiency and decreased incubation time. To the knowledge of the authors this is the first assay to use this hydrolysis enzyme for the determination of these cyclic antidepressants in a production lab. One challenge encountered during method development was the dynamic range of these analytes on this instrument. The target assay range was 1-250ng/mL, this needed to be reduced to 10-100ng/mL to accommodate the dynamic range of the instrument. This method was developed and validated to quantify patient samples in a production lab. Linearity was assessed with 5 standards across the 10-100 ng/mL range. Intra- and inter-batch (3-day) precision and accuracy assessments were performed at four levels (10, 12.5, 50, and 75 ng/mL). Inter-batch precision was less than 7.9 % (LLOQ); accuracy was within 4 % of the nominal concentration. Use of the deuterated internal standards resulted in a matrix factors between 0.98 and 1.01 for all analytes. Assessments of specificity, carryover, dilutional linearity, and impact from blood contamination (5% v/v) were performed as well as all stability assessments including freeze/thaw, short-term, long-term, post-preparative, and autosampler stability.