Valérie Thibert (Presenter)
Thermo Fisher Scientific France
Authorship: Valérie Thibert (1), Claudio De Nardi (2), Katharina Kern (3), Steffen Peters (3)
(1) Thermo Fisher Scientific, Les Ulis, France, (2) Thermo Fisher Scientific GmbH, Dreieich, Germany, (3) Recipe Chemicals+Instruments GmbH, Munich, Germany
| Short Abstract An analytical method for the quantification of 35 benzodiazepines and metabolites that allows for direct injection of human serum is reported. Conventional HPLC methods require manual offline sample preparation; in this case, internal standards are automatically added to each sample by the autosampler prior to injection of the intact serum onto an online SPE liquid chromatographer using a Thermo Scientific™ Transcend™ II system. Analytes are detected by mass spectrometry in single reaction monitoring acquisition mode on a Thermo Scientific™ TSQ Endura™ triple quadrupole with a heated electrospray ionization source. The method was analytically validated in terms of limits of quantification, linearity ranges, accuracy and precision for each analytes using the MS9050 ClinMass® TDM Platform from RECIPE with the MS9550 Add-On Set for Benzodiazepines. |
Long Abstract
Introduction
Long-term administration of benzodiazepines may require drug monitoring research. The quantification is primarily performed from serum in order to identify changes in pharmacokinetics. The analytical method reported hereby involves the automated addition of the internal standards by the autosampler prior to direct injection of intact serum onto an LC system using a Thermo Scientific™ Transcend™ II system; online solid phase extraction (SPE) allows for automated sample clean-up followed by liquid chromatography and tandem mass spectrometry detection; a Thermo Scientific™ TSQ Endura™ triple quadrupole with a heated electrospray source was used in single reaction monitoring (SRM) acquisition mode. The method, based on the MS9050 ClinMass® TDM Platform with the MS9550 Add-On Set for Benzodiazepines from RECIPE, covers 35 benzodiazepines and metabolites; a full analytical validation following international guidelines in terms of limits of quantification, linearity range, accuracy and precision was performed for 7-Aminoclonazepam, 7-Aminoflunitrazepam, 7-Aminonitrazepam, á-OH-Alprazolam, á-OH-Midazolam, á-OH-Triazolam, Alprazolam, Bromazepam, Chordiazepoxide, Clobazam, Clonazepam, Demoxepam, Desalkylflurazepam, Desmethylflunitrazepam, Diazepam, Estazolam, Flunitrazepam, Flurazepam, Lorazepam, Lormetazepam, Medazepam, Midazolam, Nitrazepam, Norclobazam, Nordiazepam, Oxazepam, Prazepam, Temazepam, Tetrazepam, Trazodone, Triazolam, Zaleplone, Zolpidem and Zopiclone.
Methods
This analytical method is based on the MS9050 ClinMass® TDM Platform with the MS9550 Add-On Set for Benzodiazepines from RECIPE and allows for the quantification of 3-Hydroxybromazepam, 7-Aminoclonazepam, 7-Aminoflunitrazepam, 7-Aminonitrazepam, á-OH-Alprazolam, á-OH-Midazolam, á-OH-Triazolam, Alprazolam, Bromazepam, Chordiazepoxide, Clobazam, Clonazepam, Demoxepam, Desalkylflurazepam, Desmethylflunitrazepam, Diazepam, Estazolam, Flunitrazepam, Flurazepam, Lorazepam, Lormetazepam, Medazepam, Midazolam, Nitrazepam, Norclobazam, Nordiazepam, Oxazepam, Prazepam, Temazepam, Tetrazepam, Trazodone, Triazolam, Zaleplone, Zolpidem and Zopiclone in human serum. Calibrators and controls at four and two different levels, respectively, are included in the kit. Twenty different deuterated internal standards are used for the quantification; these internal standards were automatically added to each sample using the autosampler of a Transcend II system prior to injection of the unextracted serum onto the LC system. Online SPE was used for automated sample clean-up followed by chromatographic separation by gradient elution on the same system using mobile phases, SPE cartridge and analytical column provided with the kit. A TSQ Endura triple quadrupole equipped with a heated electrospray source operated in positive mode was used for detection in SRM acquisition mode. Two SRM transitions were included in the acquisition method for quantification and confirmation, respectively.
Results
The assay proved to be linear for all the analytes of interest in the calibration range covered by the kit, with a correlation factor (R2) always above 0.98 and limits of quantification (LOQ) between 0.18 and 88.7 ng/mL and upper limits (ULOQ) between 27.3 ng/mL and 4.04 µg/mL. Trueness of measurement using external quality controls from Instand e.V. analyzed on four different days in a single run each day was used to evaluate assay accuracy; the percentage bias between nominal and average back-calculated concentration for these control samples was between -3.1 and 15.6 %. Repeatability was evaluated in terms of percentage coefficient of variation (%CV) using the kit controls at both levels in replicates of eight (n=8); the %CV for this intra-assay precision was always below 12.9% and 14.0% for the lower and upper control, respectively. Reproducibility was evaluated on the same controls in replicates of three (n=3) analyzed on five different days; the %CV for this inter-assay precision was always below 12.5% and 11.3% for the lower and upper control, respectively. Zopiclone was not taken into account in these evaluations due to stability issues.
Conclusions
A liquid chromatography tandem mass spectrometry method for the quantification of 35 benzodiazepines and metabolites in human serum using the MS9550 ClinMass® TDM Platform with the MS9550 Add-On Set for Benzodiazepines from RECIPE was implemented and analytically validated on a Transcend II system connected to a TSQ Endura. The method allows for automated addition of the internal standards and direct injection of intact 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 of the kit for these drugs.
References & Acknowledgements:
| Description | Y/N | Source |
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| Salary | yes | Thermo Fisher Scientific |
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| Expenses | no |
IP Royalty: no
| Planning to mention or discuss specific products or technology of the company(ies) listed above: | yes |