MSACL 2022 Abstract

Introduction/Background:
Measuring the concentrations of catecholamines, norepinephrine (NE), epinephrine (E), & dopamine (DA) from urine aids in the diagnosis of rare tumors such as pheochromocytomas and paragangliomas. It is important to be able to detect these hormones accurately and quickly. Since early diagnosis and treatment of these tumors is critical, then fast and accurate detection of these hormones is important. Existing methods using HPLC coupled with an electrochemical detector (ECD) have limitations including, specificity, long run times, & outdated unsupported instrument components. In contrast, LC-MS/MS offers several advantages including increased sensitivity & specificity, shorter run times with more advanced pump systems, and multiplexing capabilities for faster sample throughput. However, urine is a complex matrix for LC-MS/MS and requires enrichment of catecholamines to be a viable alternative to ECD. In the current study, our objective was to develop an LC-MS/MS method for analysis of catecholamines after their specific extraction from urine.

Methods:
Patient samples were prepared using an automated liquid handling system and solid phase extraction was performed using an automated positive pressure processor, all under 3 hours. Ten microliters of reconstituted urine sample was injected onto an Agilent StreamSelect LC-MS/MS system. Separation was accomplished through a reverse phase HPLC column with mobile phases consisting of acidified aqueous phase and blended organic phase. The total acquisition time was 9.75 minutes with data collection window under three minutes per injection. Validation studies included up to 120 specimen discards that were extracted and reported from the current methodology, then prepared using the testing method. Results were correlated against their original results from an ECD. Precision studies include intra-assay study of 5 replicates at 3 concentrations for each analyte, NE: 64.0 ng/mL level 1, 174.0 ng/mL level 2, 250 ng/mL level 3, E: 19.6 ng/mL level 1, 38.9 ng/mL level 2, 125.0 ng/mL, & DA: 186.0 ng/mL level 1, 264.8 ng/mL level 2, 375.0 ng/mL level 3. All levels were extracted over 5 different days for a total of 25 injections at each level. Additional precision studies include inter-assay study of 2 replicates at 3 concentrations for each analyte, NE: 42.5 ng/mL level 1, 64.0 ng/mL level 2, 174.0 ng/mL level 3, E: 12.7 ng/mL level 1, 19.6 ng/mL level 2, 38.9 ng/mL, & DA: 82.4 ng/mL level 1, 186.0 ng/mL level 2, 284.0 ng/mL level 3. All levels were extracted over 20 different days for a total of 40 injections at each level. Limit of Quantitation studies included various spiked levels of urine with known concentrations of each catecholamine. Each concentration was diluted to 5 levels, NE: 11.6 ng/mL level 5, 5.8 ng/mL level 4, 2.8 ng/mL level 3, 1.4 ng/mL level 2, 0.7 ng/mL level 1, E: 5.4 ng/mL level 5, 2.7 ng/mL level 4, 1.3 ng/mL level 3, 0.7 ng/mL level 2, 0.3 ng/mL level 1, & DA: 17.9 ng/mL level 5, 8.9 ng/mL level 4, 4.3 ng/mL level 3, 2.2 ng/mL level 2, 1.1 ng/mL level 1. All levels were extracted with 5 replicates of each level over 5 different days for a total of 25 injections. Analytical measurement range studies included 21 calibrations, at a concentration range of 2 ng/mL to 300 ng/mL, and extracted over 21 days.

Findings/Results:
Split-patient comparison results are as follows, NE: 99 points with a bias of 1.1, E: 41 points with a bias of 1.1, & DA: 90 points with a bias of 1.0. Intra-assay precision results are as follows, NE within run variation yielded 2.1% at level 1, 3.4% at level 2, & 0.9% at level 3. E within run variation yielded 5.9% at level 1, 5.5% at level 2, & 3.2% at level 3. DA within run variation yielded 1.9% at level 1, 1.7% at level 2, & 3.3% at level 3. Inter-assay precision are as follows, NE run-to-run variation yielded 4.5% at level 1, 3.4% at level 2, & 3.7% at level 3. E run-to-run variation yielded 7.8% at level 1, 7.0% at level 2, & 6.7% at level 3. DA run-to-run variation yielded 4.7% at level 1, 3.5% at level 2, & 2.7% at level 3. Limit of quantitation results are NE 2.0 ng/mL, E 1.0 ng/mL, & DA 3.0 ng/mL. Average accuracy results are NE 97.4% - 102.8%, E 93.1% - 105.1%, & DA 97.5% - 102.6%.

Conclusions:
We have developed and validated an LC-MS/MS method for urine catecholamines with automated sample preparation and high-throughput analysis.