Validation of an assay for quantification of free normetanephrine, metanephrine and methoxytyramine in plasma by high performance liquid chromatography with coulometric detection: Comparison of peak-area vs. peak-height measurements

摘要: Abstract Background Measurements of plasma concentrations free normetanephrine (NMN), metanephrine (MN) and methoxytyramine (MTY) constitute the most diagnostically accurate screening test for pheochromocytomas paragangliomas. The aim this article is to present results from a validation an analytical method utilizing high performance liquid chromatography with coulometric detection (HPLC-CD) quantifying NMN, MN MTY. Additionally, peak integration by height area use one calibration curve all batches or individual each batch samples was explored as determine optimal approach regard accuracy precision. Methods validated using charcoal stripped spiked solutions MN, MTY internal standard (4-hydroxy-3-methoxybenzylamine) exception selectivity which evaluated analysis real samples. Calibration performance, accuracy, precision recovery were determined following both peak-area peak-height measurements obtained compared. precise analyzing quality control at three concentration levels in 30 runs. Results detector response linear over entire tested range 10 2000 pg/mL R 2  ≥ 0.9988. LLOQ 10 pg/mL analyte interest. To improve low concentrations, weighted (1/amount) regression model employed, resulted inaccuracies −2.48 9.78% 0.22 7.81% integration, respectively. imprecisions ranged 1.07 15.45% 0.70 11.65% measurements, measurements. It characterized ranging −3.39 +3.27% 2.17 13.57%. Conclusions established HPLC-CD enables reasonable selectivity. Preparing based on yields