Practical liquid chromatography–tandem mass spectrometry method for the simultaneous quantification of amitriptyline, nortriptyline and their hydroxy metabolites in human serum

Abstract Amitriptyline (AMI) has been in use for decades in treating depression and more recently for the management of neuropathic pain. A highly sensitive and specific LC–tandem mass spectrometry method was developed for simultaneous determination of AMI, its active metabolite nortriptyline (NOR) and their hydroxy‐metabolites in human serum, using deuterated AMI and NOR as internal standards. The isobaric E‐10‐hydroxyamitriptyline (E‐OH AMI), Z‐10‐hydroxyamitriptyline (Z‐OH AMI), E‐10‐hydroxynortriptyline (E‐OH NOR) and Z‐10‐hydroxynortriptyline (Z‐OH NOR), together with their parent compounds, were separated on an ACE C 18 column using a simple protein precipitation method, followed by dilution and analysis using positive electrospray ionisation with multiple reaction monitoring. The total run time was 6 min with elution of E‐OH AMI, E‐OH NOR, Z‐OH AMI, Z‐OH NOR, AMI (+ deuterated AMI) and NOR (+ deuterated NOR) at 1.21, 1.28, 1.66, 1.71, 2.50 and 2.59 min, respectively. The method was validated in human serum with a lower limit of quantitation of 0.5 ng/mL for all analytes. A linear response function was established for the range of concentrations 0.5–400 ng/mL ( r 2 > .999). The practical assay was applied on samples from patients on AMI, genotyped for CYP2C19 and CYP2D6 , to understand the influence of metaboliser status and concomitant medication on therapeutic drug monitoring.