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Abstract Background: Plasma 3-methoxytyramine (3-MT), the O-methylated dopamine metabolite, is useful for detecting dopamine-producing pheochromocytomas and paragangliomas (PPGLs). Due to the difficulty in measuring the very low concentrations in plasma, its measurements have not been widely offered as part of routine workup for PPGLs. In this study, we developed a highly sensitive and specific mass spectrometric method for simultaneous measurement of plasma free metanephrine (MN), normetanephrine (NMN), and 3-MT by incorporating 3-MT measurement into previously established MN and NMN assay, and applied it to clinical practice.
Methods: All plasma samples were extracted with the solid-phase extraction (SPE) method. SPE was performed using Strata CW-X extraction cartridges (Phenomenex, Torrance, CA, USA), pretreated with 1 mL methanol, 1 mL deionized water, and 1 mL of 0.5 M ammonium acetate. Finally, 3 μL of the reconstituted eluate was injected into the liquid chromatography tandem mass spectrometry (LC-MS/MS) system. Analyses were performed on an Agilent 6490 tandem mass spectrometer equipped with an Agilent 1260 high-performance liquid chromatography (HPLC) system (Agilent Technologies, Santa Clara, CA, USA). The chromatographic separation of MN and NMN was conducted on a Unison UK C18 column (Imtakt, Portland, OR, USA; 2.0×100 mm, 3.0 μm). The analytes were detected in multiple-reaction monitoring mode by using positive electrospray ionization: MN, transition m/z 180.1 → 165.1; NMN, m/z 166.1 → 134.1; 3-MT, m/z 150.9 → 118.9). The method was validated for linearity, precision, accuracy, lower limits of quantification (LLOQ) and detection (LOD), extraction recovery, matrix effect, and carry-over according to CLSI C62-A, C64 guidelines. From August 2022 to August 2023, 3-MT was measured in clinical samples requested for MN and NMN measurement.
Results: Analytical run time was 6 min per sample and total sample preparation time was 1.5 hour per batch. Assay range and LLOQ were as follows; 0.03–50.0 nM (R²>0.99) and 0.03 nM for 3-MT; 0.04–22.0 nM (R²>0.99) and 0.04 nM for MN; 0.08–60.0 nM (R²>0.99) and 0.08 nM for NMN. The intra- and inter-day imprecisions were CV 1.1–6.9% and 2.1–9.8%, respectively. For 3-MT, accuracy was 91.5–104.0%. No interference or carryover was observed. The matrix effects ranged from 92.8% to 98.8%, and extraction recovery ranged from 96.3% to 107.2%. Among the 2,947 clinical samples, 1.4% (42/2,947) showed elevated plasma 3-MT.
Conclusions: A highly sensitive and specific LC-MS/MS method for 3-MT quantitation in plasma that could be incorporated in an established LC-MS/MS assay for metanephrines was developed and successfully applied to clinical practice. By allowing simultaneous measurement of three catecholamine metabolites, this method would thus contribute to increasing accessibility to 3-MT measurements in clinical laboratories, and provide valuable information to clinicians when diagnosing and monitoring patients with PPGL.
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= Discovery stage. (16.60%, 2024)
= Translation stage. (37.02%, 2024)
= Clinically available. (46.38%, 2024)
