Technical Note pubs.acs.org/ac
High-Throughput and Sensitive Quantitation of Plasma Catecholamines by Ultraperformance Liquid Chromatography− Tandem Mass Spectrometry Using a Solid Phase Microwell Extraction Plate Marielle Dunand,† Danilo Gubian,‡ Maxime Stauffer,† Karim Abid,† and Eric Grouzmann*,† †
Service de Biomédecine, Laboratoire des Catécholamines et Peptides and ‡Maintenance Biomédicale des Laboratoires, Centre Hospitalier Universitaire Vaudois, 1011, Lausanne, Switzerland S Supporting Information *
ABSTRACT: Plasma catecholamines provide a reliable biomarker of sympathetic activity. The low circulating concentrations of catecholamines and analytical interferences require tedious sample preparation and long chromatographic runs to ensure their accurate quantification by HPLC with electrochemical detection. Published or commercially available methods relying on solid phase extraction technology lack sensitivity or require derivatization of catecholamine by hazardous reagents prior to tandem mass spectrometry (MS) analysis. Here, we manufactured a novel 96-well microplate device specifically designed to extract plasma catecholamines prior to their quantification by a new and highly sensitive ultraperformance liquid chromatography−tandem mass spectrometry (UPLC−MS/MS) method. Processing time, which included sample purification on activated aluminum oxide and elution, is less than 1 h per 96-well microplate. The UPLC−MS/MS analysis run time is 2.0 min per sample. This UPLC−MS/MS method does not require a derivatization step, reduces the turnaround time by 10-fold compared to conventional methods used for routine application, and allows catecholamine quantification in reduced plasma sample volumes (50−250 μL, e.g., from children and mice).
P
aluminum oxide at pH 8.5 and their elution at acid pH with a yield recovery of 60−80%.6 Plasma catecholamine kits using ready-to-use solid phase extraction (SPE) tubes containing preweighted amounts of activated alumina are commercially available (Thermo Scientific; Sunnyvale, CA; Recipe GmbH; Munich, Germany; Bio-Rad, Hercules, CA; Chromsystems, Munich, Germany) but require large plasma volumes (i.e., 1 mL) and individual sample extraction procedures that increases the turn-around time of the analysis. A simple SPE extraction method for plasma catecholamines (0.3 mL) using a mixedmode nonpolar/weak cation exchange resin filled in 96 fixed plates is also available from Biotage (Uppsala, Sweden). The application note AN745 of the supplier reported acceptable lower limits of detection for the quantification of E in human plasma at 0.55 nmol/L but at the upper reference limit for NE at 5.9 nmol/L and by far exceeding the normal range (5 was 0.03 nmol/L, 0.10 nmol/L, and 0.05 nmol/L for E, NE, and DA, respectively. Table S6 in the Supporting Information summarizes regression equations, coefficients of regression and detection, and quantification limits. Method Comparison, Determination of Reference Intervals. Plasma catecholamine concentrations in plasma samples collected from patients screened for pheochromocytoma were determined by LC−MS/MS and compared with the results obtained by HPLC with electrochemical detection (Coularray system) as routinely performed in our laboratory. Deming regression showed the following curve equations: E, UPLC−MS/MS = −0.05 + 1.02; E, HPLC-ECD (n = 64); NE, UPLC−MS/MS = 0.22 + 0.92; NE, HPLC−ECD (n = 64); DA, UPLC−MS/MS = 0.15 + 0.77; DA, HPLC−ECD (n = 33) (Figure S4A−C in the Supporting Information). Bland and Altman plots for the mean difference between HPLC−ECD and UPLC−MS/MS methods were constructed and showed negative differences with mean difference of 0.03, 1.79, and 0.36 nmol/L for E, NE, and DA, respectively. NE concentrations higher than 20 nmol/L and DA concentrations higher than 3 nmol/L showed lower concentrations by UPLC− MS/MS than HPLC−ECD (Figure S5 in the Supporting Information). Additionally, we compared the catecholamine concentrations measured in seven patients having a pheochromocytoma by UPLC−MS/MS and HPLC−ECD and found similar results by both methods (Table S7 in the Supporting Information). The distribution of values was skewed for all three catecholamines, and therefore reference intervals were calculated after logarithmic transformation of the data. Reference intervals of 0.03−0.98 nmol/L for E, 0.63−4.51 nmol/L for NE, and
