Claudio De Nardi (Presenter)
Thermo Fisher Scientific
Authorship: Claudio De Nardi (1), Katharina Kern (2), Steffen Peters (2)
(1) Thermo Fisher Scientific GmbH, Dreieich, Germany (2) RECIPE Chemicals+Instruments GmbH, Munich, Germany
| Short Abstract An analytical method for the quantification of 14 tricyclic antidepressants based on direct injection of human serum is reported. The method involves automated addition of the internal standards followed by injection of the serum sample onto an online SPE liquid chromatographer using a Thermo Scientific™ Transcend™ II system. Mass spectrometric detection is performed by single reaction monitoring on a Thermo Scientific™ TSQ Endura™ triple quadrupole using heated electrospray ionization. The method was analytically validated using the MS9050 ClinMass® TDM Platform from RECIPE with the MS9150 Add-On Set for Tricyclic Antidepressants and limit of quantification, linearity range, accuracy and precision were evaluated. |
Long Abstract
Introduction
The monitoring of circulating levels of tricyclic antidepressants, mainly performed in serum, is critical to detect changes in the pharmacokinetic of the compounds. The reported analytical method is based on automated addition of the internal standards performed by the autosampler prior to direct injection of serum onto an LC system using a Thermo Scientific™ Transcend™ II system; sample clean-up is performed by online solid phase extraction (SPE) followed by liquid chromatography tandem mass spectrometry; a Thermo Scientific™ TSQ Endura™ triple quadrupole with heated electrospray ionization was used for detection by single reaction monitoring (SRM). The method, based on the MS9050 ClinMass® TDM Platform with the MS9150 Add-On Set for Tricyclic Antidepressants from RECIPE, covers 14 different tricyclic antidepressants and was analytically validated following international guidelines in terms of limits of quantification, linearity range, accuracy and precision for Amitriptyline, Clomipramine, Clozapine, Desipramine, Doxepin, Imipramine, Maprotiline, Norclomipramine, Norclozapine, Nordoxepine, Nortriptyline and Trimpramine.
Methods
This analytical method is based on the MS9050 ClinMass® TDM Platform with the MS9150 Add-On Set for Tricyclic Antidepressants from RECIPE and allows for the quantification of Amitriptyline, Imipramine, Nortrimipramine,Clomipramine, Maprotiline, Nortriptyline, Clozapine, Norclomipramine, Protriptyline,Desipramine, Norclozapine, Trimipramine, Doxepin and Nordoxepin in human serum. The kit includes calibrators and controls at four and two different levels, respectively, covering the therapeutic range for each analyte. Twelve deuterated internal standards are used for the quantification; the autosampler of a Transcend II system was used for the automated addition of these internal standards prior to injection of the unextracted serum sample onto the LC system. Sample clean-up was performed by online SPE followed by chromatographic separation by gradient elution on the same system using mobile phases, SPE cartridge and analytical column provided with the kit. Detection was performed by SRM on a TSQ Endura triple quadrupole with heated electrospray ionization operated in positive mode. Two SRM transitions were included in the acquisition method for quantification and confirmation, respectively.
Results
A full analytical validation was performed for Amitriptyline, Clomipramine, Clozapine, Desipramine, Doxepin, Imipramine, Maprotiline, Norclomipramine, Norclozapine, Nordoxepine, Nortriptyline and Trimpramine. For these analytes, the assay proved to be linear in the calibration range covered by the kit, with limits of quantification (LOQ) between 2.15 and 4.6 ng/mL, upper limits (ULOQ) between 417 and 1177 ng/mL and a correlation factor (R2) always above 0.992. Accuracy for the assay was evaluated in terms of trueness of measurement using external quality controls from Instand e.V. analyzed on four different days in a single run each day; the percentage bias between nominal and average back-calculated concentration for these control samples was between -7.2 and 17.1 %. Intra-assay precision was evaluated in terms of percentage coefficient of variation (%CV) using the kit controls at both levels in replicates of seven (n=7); the %CV for intra-assay precision was always below 4.5% and 7.2% for the lower and upper control, respectively. Inter-assay precision was evaluated on the same controls in replicates of three (n=3) analyzed on five different days; the %CV for inter-assay precision was always below 6.7% and 9.4% for the lower and upper control, respectively.
Conclusions
A liquid chromatography tandem mass spectrometry method for the quantification of a panel of 14 tricyclic antidepressants in human serum using the MS9050 ClinMass® TDM Platform from RECIPE was implemented and analytically validated on a Transcend II system connected to a TSQ Endura mass spectrometer. The method allows for automated addition of the internal standards and for direct injection of serum samples, reducing sample manipulation to a minimum. This assay proved to be compliant with international guidelines in terms of accuracy and intra- and inter-assay precision and to have the sensitivity and linearity of response suitable to cover the concentration range required by the kit for these drugs.
References & Acknowledgements:
| Description | Y/N | Source |
| Grants | no | |
| Salary | yes | Thermo Fisher Scientific |
| Board Member | no | |
| Stock | no | |
| Expenses | no |
IP Royalty: no
| Planning to mention or discuss specific products or technology of the company(ies) listed above: | yes |