Marta Kozak (Presenter)
ThermoFisher Scientific
Bio: Marta Kozak is employed by Thermo Scientific LS-MS division in San Jose, California. She is an application group manager working on clinical research, toxicology and drugs of abuse analytical methods. She has MS degree in analytical chemistry and has 25 years experience in analytical methods development, validation and research in chemical, pharmaceutical, clinical research and toxicology applications.
Authorship: Mindy Gao, Marta Kozak
Thermo Fisher Scientific
| Short Abstract We evaluated an analytical method to support analysis of catecholamines (epinephrine, norepinephrine and dopamine) in human plasma for research. The method used SPE for sample preparation, a 9 min gradient chromatographic separation and a triple quadrupole mass spectrometer collecting two SRM for each analyte to calculate ion ratio. The limit of quantitation in donor plasma was 5 pg/mL for dopamine and 25 pg/mL for epinephrine and norepinephrine. Method precision obtained for RECIPETM QC samples was better than 4.6% and accuracy ranged from 86.6-119%. Ionization suppression was observed only for epinephrine and it was corrected by deuterated internal standard. |
Long Abstract
Introduction
We evaluated an analytical method to support the analysis of catecholamines (epinephrine, norepinephrine and dopamine) in human plasma for research. The method used SPE for sample preparation, a 9 min gradient for chromatographic separation and a triple quadrupole mass spectrometer for analyte detection. The use of calibration standards prepared in solvent without SPE processing, calibration standards prepared in solvent with SPE processing and calibration standards prepared in analyte-free human plasma with SPE processing was evaluated. Method performance was characterized by using RECIPE QC samples and in-house prepared spiked donor plasma samples.
Methods
A 500-µL aliquot of plasma sample was mixed with 500 µL of a buffer containing internal standards and processed through a BiotageTM EVOLUTETM Express WCX 10mg 96-well plate with the developed method. A 15-µL aliquot of eluted sample was injected onto a Restek UltraTM PFPP 150x2.1mm, 3µm column with a 9-minute method to separate endogenous plasma components from the analytes of interest. Detection was performed with a Thermo Scientific™ TSQ QuantivaTM mass spectrometer equipped with HESI ionization source. Several SRM transitions for each analyte were evaluated and the two most robust SRM transitions for each analyte were selected to calculate ion ratio. The method performance evaluation included: limit of quantitation (LOQ), linearity range, intra-batch and inter-batch accuracies and precisions, matrix effects and stability of processed samples at -30 deg C.
Results
The limit of quantitation in donor plasma was 5 pg/mL for dopamine and 25 pg/mL for epinephrine and norepinephrine. The upper calibration range was 1000 pg/mL for dopamine and 5000 pg/mL for epinephrine and norepinephrine. Intra-batch precision was better than 2.8% and inter-batch precision was better than 4.6%. The accuracy ranged from 86.6-119% and 88.0-111% for Level I and Level II QC samples, respectively. Negligible matrix effects were observed for dopamine and norepinephrine. The average peak area recovery in six spiked donor plasma was 92.9% for dopamine and 84.8% for norepinephrine. Ionization suppression was observed for epinephrine. The peak area recovery was 24.6%, and it was corrected for by the deuterated analog internal standard.
Accurate results were obtained for QC samples using all three types of calibrators indicating that any of the three methodologies investigated for calibration curve processing can be used in a quantitative analysis.
Conclusions
Sensitive and robust analyses of catecholamines in plasma were demonstrated. Three different calibration curve processing techniques all give accurate results for QC samples. Matrix effects are only observed for one of the compounds analyzed and are adequately corrected for by use of its deuterated analog as an internal standard. The analytical throughput can be improved by implementing the method on multi-channel LC system since data acquisition window is only about 25% of the total run time.
References & Acknowledgements:
| Description | Y/N | Source |
| Grants | no | |
| Salary | yes | ThermoFisher 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 |