| Article ID | Journal | Published Year | Pages | File Type |
|---|---|---|---|---|
| 1212056 | Journal of Chromatography B | 2015 | 8 Pages |
•The assay enables measurements of free metanephrines and methoxytyramine in plasma.•Analytes of interest are measured from 10 pg/mL with good accuracy and precision.•Everyday calibration using peak heights provided the best accuracy and precision.
BackgroundMeasurements of plasma concentrations of free normetanephrine (NMN), metanephrine (MN) and methoxytyramine (MTY) constitute the most diagnostically accurate screening test for pheochromocytomas and paragangliomas. The aim of this article is to present the results from a validation of an analytical method utilizing high performance liquid chromatography with coulometric detection (HPLC-CD) for quantifying plasma free NMN, MN and MTY. Additionally, peak integration by height and area and the use of one calibration curve for all batches or individual calibration curve for each batch of samples was explored as to determine the optimal approach with regard to accuracy and precision.MethodsThe method was validated using charcoal stripped plasma spiked with solutions of NMN, MN, MTY and internal standard (4-hydroxy-3-methoxybenzylamine) with the exception of selectivity which was evaluated by analysis of real plasma samples. Calibration curve performance, accuracy, precision and recovery were determined following both peak-area and peak-height measurements and the obtained results were compared. The most accurate and precise method of calibration was evaluated by analyzing quality control samples at three concentration levels in 30 analytical runs.ResultsThe detector response was linear over the entire tested concentration range from 10 to 2000 pg/mL with R2 ≥ 0.9988. The LLOQ was 10 pg/mL for each analyte of interest. To improve accuracy for measurements at low concentrations, a weighted (1/amount) linear regression model was employed, which resulted in inaccuracies of −2.48 to 9.78% and 0.22 to 7.81% following peak-area and peak-height integration, respectively. The imprecisions ranged from 1.07 to 15.45% and from 0.70 to 11.65% for peak-area and peak-height measurements, respectively. The optimal approach to calibration was the one utilizing an individual calibration curve for each batch of samples and peak-height measurements. It was characterized by inaccuracies ranging from −3.39 to +3.27% and imprecisions from 2.17 to 13.57%.ConclusionsThe established HPLC-CD method enables accurate and precise measurements of plasma free NMN, MN and MTY with reasonable selectivity. Preparing calibration curve based on peak-height measurements for each batch of samples yields optimal accuracy and precision.
