In Matrix Derivatization Combined with LC-MS/MS Results in Ultrasensitive Quantification of Plasma Free Metanephrines and Catecholamines

Abstract

Plasma-free metanephrines and catecholamines are essential markers in the biochemical diagnosis and follow-up of neuroendocrine tumors and inborn errors of metabolism. However, their low circulating concentrations (in the nanomolar range) and poor fragmentation characteristics hinder facile simultaneous quantification by liquid chromatography and tandem mass spectrometry (LC-MS/MS). Here, we present a sensitive and simple matrix derivatization procedure using propionic anhydride that enables simultaneous quantification of unconjugated l-DOPA, catecholamines, and metanephrines in plasma by LC-MS/MS. Dilution of propionic anhydride 1:4 (v/v) in acetonitrile in combination with 50 μL of plasma resulted in the highest mass spectrometric response. In plasma, derivatization resulted in stable derivatives and increased sensitivity by a factor of 4-30 compared with a previous LC-MS/MS method for measuring plasma metanephrines in our laboratory. Furthermore, propionylation increased specificity, especially for 3-methoxytyramine, by preventing interference from antihypertensive medication (β-blockers). The method was validated according to international guidelines and correlated with a hydrophilic interaction LC-MS/MS method for measuring plasma metanephrines (R² > 0.99) and high-performance liquid chromatography with an electrochemical detection method for measuring plasma catecholamines (R² > 0.85). Reference intervals for l-DOPA, catecholamines, and metanephrines in n = 115 healthy individuals were established. Our work shows that analytes in the subnanomolar range in plasma can be derivatized in situ without any preceding sample extraction. The developed method shows improved sensitivity and selectivity over existing methods and enables simultaneous quantification of several classes of amines.

Figure 1

Effect of different derivatization reaction conditions on the internal standard peak-area response for the three different catecholamines. Internal standard peak area is shown on the y axis, and ratio of propionic anhydride to acetonitrile (v/v) is on the x axis. (A) Results for dopamine-d₄ for 50 and 100 μL of plasma. (B) Results for norepinephrine-d₆ for 50 and 100 μL of plasma. (C) Results for epinephrine-d₃ for 50 and 100 μL of plasma. (D) Results for 3-methoxytyramine-d₄ for 50 and 100 μL of plasma. (E) Results for normetanephrine-d₃ for 50 and 100 μL of plasma. (F) Results for metanephrine-d₃ for 50 and 100 μL of plasma. Results for l-DOPA-d₃ are shown in Figure S-4.

Figure 2

Derivatization reaction of dopamine with propionic anhydride. Formed derivative product is shown on the right side.

Figure 3

Proposed fragmentation scheme for each analyte: (A) l-DOPA; (B) dopamine; (C) norepinephrine; (D) epinephrine; (E) 3-methoxytyramine; (F) normetanephrine. Scheme for metanephrine is shown in Figure S-10.

Figure 4

LC-MS/MS analyses of plasma from a healthy volunteer (A), from one patient with pheochromocytoma (B1/B2), and from one patient with HNPGL (C) in the SRM mode. Chromatograms were normalized to the same intensity. (A) Calculated concentrations in plasma were as follows: l-DOPA, 12 nmol/L; DA, 0.063 nmol/L; NE, 2.3 nmol/L; E, 0.20 nmol/L; 3-MT, 0.010 nmol/L; NMN, 0.47 nmol/L; MN, 0.16 nmol/L. Calculated concentrations in plasma were (for B and C, respectively) as follows: l-DOPA, 38 and 11 nmol/L; DA, 0.26 and 1.3 nmol/L; NE, 5.2 and 5.9 nmol/L; E, 5.4 and 0.14 nmol/L; 3-MT, 0.033 and 0.22 nmol/L; NMN, 2.5 and 0.67 nmol/L; MN, 5.2 and 0.084 nmol/L. Abbreviations: DA, dopamine; NE, norepinephrine; E, epinephrine; 3-MT, 3-methoxytyramine; NMN, normetanephrine; MN, metanephrine; HNPGL, head and neck paraganglioma.